A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

Background

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.

Results

We analyzed the immunological consequences of the interaction of MHC class II (I-A^u) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4⁺ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation.     

Induction of human immunodeficiency virus (HIV-1) envelope specific cell-mediated immunity by a non-homologous synthetic peptide

Background

Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.

Methodology/Principal Findings

The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.

Conclusions/Significance

For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.     

Increased CD4+/CD8+ double-positive T cells in chronic Chagasic patients

Background

CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out.

Methodology/Principal Findings

Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor.

Conclusions

Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

Background

Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable.

Methodology/Principal Finding

In the present large-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly abundant mRNA were found to be much more likely to be the source of MHC ligands. Of the 2.5% most abundant mRNAs as much as 41% of the proteins encoded by these mRNAs contained MHC class I ligands. For proteins containing MHC class II ligands, the corresponding percentage was 11%. Furthermore, we found that most proteins containing MHC class I ligands were localised to the intracellular parts of the cell including the cytoplasm and nucleus. MHC class II ligand donors were, on the other hand, mostly membrane proteins.

Conclusions/Significance

The results contribute to the ongoing debate concerning the nature of MHC ligand-containing proteins and can be used to extend the existing methods for MHC ligand predictions by including the source protein''s localisation and expression profile. Improving the current methods is important in the growing quest for epitopes that can be used for vaccine or diagnostic purposes, especially when it comes to large DNA viruses and cancer.     

The Leukocyte Immunoglobulin-Like Receptor Family Member LILRB5 Binds to HLA-Class I Heavy Chains

Objective

The leukocyte immunoglobulin-like receptor (LILR) family includes inhibitory and stimulatory members which bind to classical and non-classical HLA-class I. The ligands for many LILR including LILRB5 have not yet been identified.

Methods

We generated C-terminal eGFP and N-terminal FLAG-tagged fusion constructs for monitoring LILR expression. We screened for LILR binding to HLA-class I by tetramer staining of 293T cells transfected with LILRA1, A4, A5 A6 and LILRB2 and LILRB5. We also studied HLA class I tetramer binding to LILRB5 on peripheral monocyte cells. LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation.

Results

HLA-B27 (B27) free heavy chain (FHC) dimer but not other HLA-class I stained LILRB5-transfected 293T cells. B27 dimer binding to LILRB5 was blocked with the class I heavy chain antibody HC10 and anti-LILRB5 antisera. B27 dimers also bound to LILRB5 on peripheral monocytes. HLA-B7 and B27 heavy chains co-immunoprecipitated with LILRB5 in transduced B and rat basophil RBL cell lines.

Conclusions

Our findings show that class I free heavy chains are ligands for LILRB5. The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to β2m-associated HLA-class I.     

Natural Killer Cell Tolerance Persists Despite Significant Reduction of Self MHC Class I on Normal Target Cells in Mice

Background

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8⁺ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.

Methodology/Principal Findings

In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.

Conclusion

Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.     

The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background

Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known.

Methodology/Principal Findings

Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation.

Significance

Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.     

An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer.

A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response. Our research has focused on developing the purification process for preparing tetramer reagent. Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable. In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration. The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC. The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step. The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate. The tetramer is further purified to remove any excess MHC or avidin components. Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process.     

The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes.

Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRalphabeta and TCRgammadelta CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3(b)-TL and murine beta(2)-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3(b)-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8(+) intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8(+) and CD4(+)CD8(+) thymocytes, were stained, while only very minor populations of CD8(+) cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3(b)-TL tetramers to CD8(+) iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3(b)-TL tetramers. In addition, CD3-independent T3(b)-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer(+)CD8(+) T lymphocytes.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Systematic Analysis of Immune Infiltrates in High-Grade Serous Ovarian Cancer Reveals CD20, FoxP3 and TIA-1 as Positive Prognostic Factors

Background

Tumor-infiltrating T cells are associated with survival in epithelial ovarian cancer (EOC), but their functional status is poorly understood, especially relative to the different risk categories and histological subtypes of EOC.

Methodology/Principal Findings

Tissue microarrays containing high-grade serous, endometrioid, mucinous and clear cell tumors were analyzed immunohistochemically for the presence of lymphocytes, dendritic cells, neutrophils, macrophages, MHC class I and II, and various markers of activation and inflammation. In high-grade serous tumors from optimally debulked patients, positive associations were seen between intraepithelial cells expressing CD3, CD4, CD8, CD45RO, CD25, TIA-1, Granzyme B, FoxP3, CD20, and CD68, as well as expression of MHC class I and II by tumor cells. Disease-specific survival was positively associated with the markers CD8, CD3, FoxP3, TIA-1, CD20, MHC class I and class II. In other histological subtypes, immune infiltrates were less prevalent, and the only markers associated with survival were MHC class II (positive association in endometrioid cases) and myeloperoxidase (negative association in clear cell cases).

Conclusions/Significance

Host immune responses to EOC vary widely according to histological subtype and the extent of residual disease. TIA-1, FoxP3 and CD20 emerge as new positive prognostic factors in high-grade serous EOC from optimally debulked patients.     

Amino acid similarity accounts for T cell cross-reactivity and for "holes" in the T cell repertoire

Background

Cytotoxic T cell (CTL) cross-reactivity is believed to play a pivotal role in generating immune responses but the extent and mechanisms of CTL cross-reactivity remain largely unknown. Several studies suggest that CTL clones can recognize highly diverse peptides, some sharing no obvious sequence identity. The emerging realization in the field is that T cell receptors (TcR) recognize multiple distinct ligands.

Principal Findings

First, we analyzed peptide scans of the HIV epitope SLFNTVATL (SFL9) and found that TCR specificity is position dependent and that biochemically similar amino acid substitutions do not drastically affect recognition. Inspired by this, we developed a general model of TCR peptide recognition using amino acid similarity matrices and found that such a model was able to predict the cross-reactivity of a diverse set of CTL epitopes. With this model, we were able to demonstrate that seemingly distinct T cell epitopes, i.e., ones with low sequence identity, are in fact more biochemically similar than expected. Additionally, an analysis of HIV immunogenicity data with our model showed that CTLs have the tendency to respond mostly to peptides that do not resemble self-antigens.

Conclusions

T cell cross-reactivity can thus, to an extent greater than earlier appreciated, be explained by amino acid similarity. The results presented in this paper will help resolving some of the long-lasting discussions in the field of T cell cross-reactivity.     

Dendritic cells reveal a broad range of MHC class I epitopes for HIV-1 in persons with suppressed viral load on antiretroviral therapy

Background

HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8⁺ T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8⁺ T cells, and could potentially be targeted to activate memory CD8⁺ T cells to a broad array of HIV-1 epitopes during ART.

Principal Findings

We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8⁺ T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a.

Significance

There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8⁺ T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.     

iNKT Cells Control Mouse Spontaneous Carcinoma Independently of Tumor-Specific Cytotoxic T Cells

Background

CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression.

Principal Findings

We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate.

Conclusions

Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.     

HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

Background

Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.

Methods

In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4⁺ T-cell lines derived from RV144 volunteers.

Results

Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4⁺ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.

Conclusions

Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4⁺T cell and antibody responses in the RV144 vaccinees.     

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

Introduction

The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes.

Objective

To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes.

Methods

A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides.

Results

We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4⁺ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4⁺ T cell expansion.

Conclusions

We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.     

Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants

Background

Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected.

Methodology/Principal Findings

We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients'' natural killer (NK) cells were depleted prior to cell transplantation.

Conclusions/Significance

Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.     

L925I Mutation in the Para-Type Sodium Channel Is Associated with Pyrethroid Resistance in Triatoma infestans from the Gran Chaco Region

Background

Chagas'' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil.The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species.

Methods and Findings

In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field.

Conclusions and Significance

The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas'' disease transmission.     

Development of a Novel In Silico Docking Simulation Model for the Fine HIV-1 Cytotoxic T Lymphocyte Epitope Mapping

Introduction

Class I HLA''s polymorphism has hampered CTL epitope mapping with laborious experiments. Objectives are 1) to evaluate the novel in silico model in predicting previously reported epitopes in comparison with existing program, and 2) to apply the model to predict optimal epitopes with HLA using experimental results.

Materials and Methods

We have developed a novel in silico epitope prediction method, based on HLA crystal structure and a peptide docking simulation model, calculating the peptide-HLA binding affinity at four amino acid residues in each terminal. It was applied to predict 52 HIV best–defined CTL epitopes from 15-mer overlapping peptides, and its predictive ability was compared with the HLA binding motif-based program of HLArestrictor. It was then used to predict HIV-1 Gag optimal epitopes from previous ELISpot results.

Results

43/52 (82.7%) epitopes were detected by the novel model, whereas 37 (71.2%) by HLArestrictor. We also found a significant reduction in epitope detection rates for longer epitopes in HLArestrictor (p = 0.027), but not in the novel model. Improved epitope prediction was also found by introducing both models, especially in specificity (p<0.001). Eight peptides were predicted as novel, immunodominant epitopes in both models.

Discussion

This novel model can predict optimal CTL epitopes, which were not detected by an existing program. This model is potentially useful not only for narrowing down optimal epitopes, but predicting rare HLA alleles with less information. By introducing different principal models, epitope prediction will be more precise.