Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition

Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.     

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.     

Cross-reactivity of T-cell clones specific for altered peptide ligands of myelin basic protein

We have determined that certain altered peptide ligands (APLs) can induce T-cells specific for the native peptide myelin basic protein (MBP) p85-99 to secrete Th2-type cytokines such as IL-4 and IL-5 in the absence of significant Th1-type cytokines. However, it is not known whether stimulation with APLs will activate autoreactive T cells or a distinct population of cells. In the present study, 18 T-cell clones that reacted with either MBP p85-99 or one of three APLs of the peptide substituted at TCR contact residues were generated. T-cells were tested functionally for their reactivity to the original stimulating peptide as well as to the MBP APLs. In addition, the T-cell receptor (TCR) alpha and beta chains of each of these clones were sequenced. In a series of T-cell clones isolated from a multiple sclerosis patient, stimulation of T-cells with the APL 93A, which has an alanine for lysine substitution at the TCR contact residue 93, did not induce substantial proliferation of MBPp85-99-specific T-cell clones, indicating that a distinct set of T-cell clones was induced. However, this was not the case for another set of T-cell clones from a different individual in which the 93A peptide induced clonal expansion of T-cells highly reactive with the native MBPp85-99 antigen. Thus, the potential beneficial effect of using APLs to induce downregulatory cytokines appears to depend on the specific T-cell repertoire of the individual patient.     

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide

Introduction

Immunosuppressants, including anti-TNFα antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis.

Methods

DBA/1 mice were immunized with hGPI_325-339, and cells of draining lymph node (DLN) were stimulated with hGPI_325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4⁺ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI_325-339 were synthesized. CD4⁺ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI_325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI_325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA.

Results

Human GPI_325-339-specific Th17 cells showed predominant usage of TCRVβ8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4⁺ T cells in the presence of hGPI_325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL.

Conclusions

We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.     

Fast computing betweenness centrality with virtual nodes on large sparse networks

Betweenness centrality is an essential index for analysis of complex networks. However, the calculation of betweenness centrality is quite time-consuming and the fastest known algorithm uses O(N(M + N log N)) time and O(N + M) space for weighted networks, where N and M are the number of nodes and edges in the network, respectively. By inserting virtual nodes into the weighted edges and transforming the shortest path problem into a breadth-first search (BFS) problem, we propose an algorithm that can compute the betweenness centrality in O(wDN2) time for integer-weighted networks, where w is the average weight of edges and D is the average degree in the network. Considerable time can be saved with the proposed algorithm when w < log N/D + 1, indicating that it is suitable for lightly weighted large sparse networks. A similar concept of virtual node transformation can be used to calculate other shortest path based indices such as closeness centrality, graph centrality, stress centrality, and so on. Numerical simulations on various randomly generated networks reveal that it is feasible to use the proposed algorithm in large network analysis.     

An altered self-peptide with superagonist activity blocks a CD8-mediated mouse model of type 1 diabetes

T cell tolerance can be experimentally induced through administration of self-peptides with single amino acid substitution (altered peptide ligands or APLs). However, little is known about the effects of APLs on already differentiated autoreactive CD8+ T cells that play a pivotal role in the pathogenesis of autoimmune diabetes. We generated a panel of APLs derived from an influenza virus hemagglutinin peptide exhibiting in vitro functions ranging from antagonism to superagonism on specific CD8+ T cells. A superagonist APL was further characterized for its therapeutic activity in a transgenic mouse model of type 1 diabetes. When injected i.v. 1 day after the transfer of diabetogenic hemagglutinin-specific CD8+ T cells into insulin promoter-hemagglutinin transgenic mice, the superagonist APL proved more effective than the native hemagglutinin peptide in blocking diabetes. This protective effect was associated with an inhibition of CD8+ T cell cytotoxicity in vivo and with a decreased accumulation of these cells in the pancreas, leading to a marked reduction of intrainsulitis. In conclusion, a superagonist "self-peptide" APL was more effective than the native peptide in treating a CD8+ T cell-mediated diabetes model.     

PIC-1/SUMO-1-Modified PML-Retinoic Acid Receptor α Mediates Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia

Fusion proteins involving the retinoic acid receptor alpha (RARalpha) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha, not the PLZF-RARalpha, fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha-positive) APLs with As2O3 will not be successful.     

Nonparametric sparsification of complex multiscale networks

Many real-world networks tend to be very dense. Particular examples of interest arise in the construction of networks that represent pairwise similarities between objects. In these cases, the networks under consideration are weighted, generally with positive weights between any two nodes. Visualization and analysis of such networks, especially when the number of nodes is large, can pose significant challenges which are often met by reducing the edge set. Any effective "sparsification" must retain and reflect the important structure in the network. A common method is to simply apply a hard threshold, keeping only those edges whose weight exceeds some predetermined value. A more principled approach is to extract the multiscale "backbone" of a network by retaining statistically significant edges through hypothesis testing on a specific null model, or by appropriately transforming the original weight matrix before applying some sort of threshold. Unfortunately, approaches such as these can fail to capture multiscale structure in which there can be small but locally statistically significant similarity between nodes. In this paper, we introduce a new method for backbone extraction that does not rely on any particular null model, but instead uses the empirical distribution of similarity weight to determine and then retain statistically significant edges. We show that our method adapts to the heterogeneity of local edge weight distributions in several paradigmatic real world networks, and in doing so retains their multiscale structure with relatively insignificant additional computational costs. We anticipate that this simple approach will be of great use in the analysis of massive, highly connected weighted networks.     

Her-2/<Emphasis Type="Italic">neu</Emphasis> altered peptide ligand–induced CTL responses: implications for peptides with increased HLA affinity and T-cell-receptor interaction

In this study, we developed two Her-2/neu-derived E75 altered peptide ligands (APLs) that demonstrate increased affinities for the HLA-A*0201 allele compared with wild-type E75 peptide. The APLs contain amino acids from E75_(369–377), an immunodominant Her-2/neu-derived peptide, and preferred primary and auxiliary HLA-A*0201 molecule anchor residues previously identified from combinatorial peptide library screening with the recombinant molecule. CTL lines were generated against wild-type E75 peptide (KIFGSLAFL) and APLs by multiple rounds of peptide stimulation of peripheral blood mononuclear cells (PBMCs) from HLA-A2⁺ antigen normal individuals. CTL lines raised on wild-type E75 peptide cross-reacted with APLs and similarly, CTL lines raised on APLs cross-reacted with wild-type E75 peptide, as measured by IFN- ELISpot and target cell lysis assays. One of five individuals demonstrated specificity for APL 2 (FLFGSLAFL), whereas APL 5 (FLFESLAFL)-specific responses were observed from all five individuals tested. Molecular models of the E75, APL 2, and APL 5/HLA-A2 complexes indicated that the substitution of glycine with glutamic acid at position four of APL 5 resulted in the presentation of a large, negatively charged side chain that interacts with the outer edge of the HLA-A2 antigen alpha helix and is freely available to interact with cognate T-cell receptors. The results of this study further substantiate the concept that rational design of T-cell epitopes may lead to stronger peptide immunogens than natural, wild-type peptides.     

Emergence of scale-free distribution in protein–protein interaction networks based on random selection of interacting domain pairs

Recent analyses of biological and artificial networks have revealed a common network architecture, called scale-free topology. The origin of the scale-free topology has been explained by using growth and preferential attachment mechanisms. In a cell, proteins are the most important carriers of function, and are composed of domains as elemental units responsible for the physical interaction between protein pairs. Here, we propose a model for protein–protein interaction networks that reveals the emergence of two possible topologies. We show that depending on the number of randomly selected interacting domain pairs, the connectivity distribution follows either a scale-free distribution, even in the absence of the preferential attachment, or a normal distribution. This new approach only requires an evolutionary model of proteins (nodes) but not for the interactions (edges). The edges are added by means of random interaction of domain pairs. As a result, this model offers a new mechanistic explanation for understanding complex networks with a direct biological interpretation because only protein structures and their functions evolved through genetic modifications of amino acid sequences. These findings are supported by numerical simulations as well as experimental data.     

Hierarchical ordering of reticular networks

The structure of hierarchical networks in biological and physical systems has long been characterized using the Horton-Strahler ordering scheme. The scheme assigns an integer order to each edge in the network based on the topology of branching such that the order increases from distal parts of the network (e.g., mountain streams or capillaries) to the "root" of the network (e.g., the river outlet or the aorta). However, Horton-Strahler ordering cannot be applied to networks with loops because they they create a contradiction in the edge ordering in terms of which edge precedes another in the hierarchy. Here, we present a generalization of the Horton-Strahler order to weighted planar reticular networks, where weights are assumed to correlate with the importance of network edges, e.g., weights estimated from edge widths may correlate to flow capacity. Our method assigns hierarchical levels not only to edges of the network, but also to its loops, and classifies the edges into reticular edges, which are responsible for loop formation, and tree edges. In addition, we perform a detailed and rigorous theoretical analysis of the sensitivity of the hierarchical levels to weight perturbations. In doing so, we show that the ordering of the reticular edges is more robust to noise in weight estimation than is the ordering of the tree edges. We discuss applications of this generalized Horton-Strahler ordering to the study of leaf venation and other biological networks.     

Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263-275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.     

Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

Lipid Efflux Mediated by Alkylphospholipids in HepG2 Cells

Antitumoural alkylphospholipid (APL) analogues alter cholesterol homoeostasis in HepG2 cells by interfering with cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and at the same time stimulating the release of considerable quantities of membrane cholesterol. The capacity of APLs to stimulate cholesterol efflux is suppressed when cells are incubated simultaneously with APLs and serum whilst the inhibition of cholesterol transport to the ER (measured in terms of the synthesis of esterified cholesterol) persists, indicating that both effects are independent of each other. Interestingly, our results suggest that both raft and non-raft membrane domains contribute to the cholesterol released to APLs. In addition, a marked efflux of choline-bearing phospholipids (phosphatidylcholine (PC) and sphingomyelins (SM)) was found to be related to this release of cholesterol. Finally, we observed that APL micelles composed of cholesterol might act as donor/acceptor cholesterol systems. Thus, the findings of this study clearly demonstrate that antitumoural APLs act as extracellular acceptors, stimulating cholesterol and phospholipid efflux, although they may also play a role as cholesterol donors.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.