Class Of Molecules Research Articles

Crystal structure of suboptimal viral fragments of Epstein Barr Virus Rta peptide-HLA complex that stimulate CD8 T cell response

Peptides presented by Human leukocyte antigen (HLA) class-I molecules are generally 8–10 amino acids in length. However, the predominant pool of peptide fragments generated by proteasomes is less than 8 amino acids in length. Using the Epstein - Barr virus (EBV) Rta-epitope (ATIGTAMYK, residues 134–142) restricted by HLA-A*11:01 which generates a strong immunodominant response, we investigated the minimum length of a viral peptide that can constitute a viral epitope recognition by CD8 T cells. The results showed that Peripheral blood mononuclear cells (PBMCs) from healthy donors can be stimulated by a viral peptide fragment as short as 4-mer (AMYK), together with a 5-mer (ATIGT) to recapitulate the full length EBV Rta epitope. This was confirmed by generating crystals of the tetra-complex (2 peptides, HLA and β2-microglobulin). The solved crystal structure of HLA-A*11:01 in complex with these two short peptides revealed that they can bind in the same orientation similar to parental peptide (9-mer) and the free ends of two short peptides acquires a bulged conformation that is directed towards the T cell receptor. Our data shows that suboptimal length of 4-mer and 5-mer peptides can complement each other to form a stable peptide-MHC (pMHC) complex.

The Dendritic Cell HLA-Class-II/Therapeutic Factor VIII (FVIII) Peptidome Is Influenced in Unanticipated Ways By the B-Domain of FVIII and the FVIII Chaperon Protein, Von Willebrand Factor: The Outrigger and Glycosylation-Umbrella (GUMB) Hypotheses

Background: Patients with the X-linked bleeding disorder hemophilia A have impaired blood clotting due to deficient or absent factor VIII (FVIII) coagulant activity. Bleeding can be managed by infusions of any of several plasma-derived (pd) or recombinant (r) therapeutic FVIII proteins (tFVIIIs). The efficacy of tFVIIIs can be eliminated, however, if neutralizing anti-tFVIII-antibodies called "inhibitors" develop. Since the development of inhibitors is T-helper-cell dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules comprise an important early determinant. Accumulating evidence suggests that the presence of the FVIII chaperon protein, von Willebrand factor (VWF), in either pdFVIII or rFVIII concentrates, decreases the immunogenicity of these tFVIIIs by reducing their uptake by antigen presenting cells, especially dendritic cells (DCs). Objectives: Use a native (i.e., non-engineered) full-length (FL) tFVIII without ((−)) or with ((+)) pdVWF in DC-protein processing and presentation assays (PPPAs) followed by mass-spectrometric and peptide-proteomic analyses to identify and quantify the DP-, DQ-, and DR-bound/tFVIII-derived-peptides in individual HLAcII repertoires. Compare the number of peptides in the subset from any of the five globular domains (A1, A2, A3, C1 and C2) or three acidic-residue-rich connecting segments (a1, a2 and a3), which we collectively refer to as the non-B-domain (NBD) portion of a tFVIII, with the number of peptides from its non-globular "outrigger like" B-domain (BD) that contains ~80% of the N-linked glycans of a FL-FVIII molecule despite containing only 908 amino acid residues, i.e. slightly less than 40% of its 2,332 total residues. Methods: DC-PPPAs were performed using monocyte-derived (Mo)DCs obtained from 12 healthy blood donors. The tFVIII tested was a FL-rFVIII (Advate®) used (−) or (+) pdVWF (i.e., FL-rFVIII − pdVWF and FL-rFVIII + pdVWF) and the resulting data consists of counts of tFVIII-derived peptides presented on and extracted from HLAcII molecules. Difference of proportion tests were used to compare the effect of pdVWF as well as the NBD- and BD-regions of the FL tFVIII on the peptide counts. Results: FL-rFVIII − pdVWF yielded significantly more peptides (p<0.05) than FL-rFVIII + pdVWF from the NBD portions but not the BD. Interestingly, for the FL-rFVIII − pdVWF preparation, the NBD portions yielded significantly more peptides (p<0.05) than the BD, but this pattern was reversed for the FL-rFVIII + pdVWF preparation in that the NBD portions yielded significantly less peptides (p<0.05) than the BD. Conclusions: The Outrigger Hypothesis posits that in the presence of VWF the heavily glycosylated BD acts as an "outrigger" and renders this portion of a FL tFVIII relatively more likely to be internalized, proteolytically processed and HLAcII-presented. However, in the absence of VWF, the N-linked glycans individually act to protect the amide bonds in the underlying peptide bond backbone of a tFVIII from proteolytic processing, as posited by the GUMB Hypothesis. Our results support both hypotheses as important determinants in the pathogenesis of inhibitor development. Disclosures Howard: Haplogenics Corporation: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Luu:Haplogenics Corporation: Employment. Hofmann:CSL Behring: Employment. Dinh:Haplogenics Corporation: Employment. Mead:CSL Behring: Employment. Powell:Haplogenics: Membership on an entity's Board of Directors or advisory committees. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees. Eugene:CSL Behring: Employment.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications.

Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder - nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

A Molecularly Detailed NaV1.5 Model Reveals a New Class I Antiarrhythmic Target

Antiarrhythmic treatment strategies remain suboptimal due to our inability to predict how drug interactions with ion channels will affect the ability of the tissues to initiate and sustain an arrhythmia. We built a multiscale molecular model of the Na+ channel domain III (domain III voltage-sensing domain) to highlight the molecular underpinnings responsible for mexiletine drug efficacy. This model predicts that a hyperpolarizing shift in the domain III voltage-sensing domain is critical for drug efficacy and may be leveraged to design more potent ClassI molecules. The model was therefore used to design, in silico, a theoretical mexiletine booster that can dramatically rescue a mutant resistant to the potent antiarrhythmic effects of mexiletine. Our framework provides a strategy for in silico design of precision-targeted therapeutic agents that simultaneously assesses antiarrhythmic markers of success and failure at multiple spatial and time scales. This approach provides a roadmap for the design of novel molecular-based therapy to treat myriad arrhythmia syndromes, includingventricular tachycardia, heart failure arrhythmias, and inherited arrhythmia syndromes.

CD8+ T Lymphocyte and NK Cell Network: Circuitry in the Cytotoxic Domain of Immunity.

Multiple effector layers in the immune system ensure an optimal temporal and spatial distribution of immune defense. Cytotoxic innate lymphoid natural killers (NK) and adaptive CD8+ T lymphocytes (CTL) interact to elicit specific cytolytic outcomes. The CTL carry antigen-specific T cell receptors (TCR) to recognize cognate peptides bound with major histocompatibility complex class-I (MHC-I) or human leukocyte antigen (HLA) molecules on target cells. Upon TCR engagement with MHC-I:peptide at a threshold of avidity, T cell intracellular programs converge into cytolytic activity. By contrast, NK cells lack antigen-specific receptors but express a repertoire of highly polymorphic and polygenic inhibitory and activating receptors that bind various ligands including MHC and like molecules. A highly calibrated maturation enables NK cells to eliminate target cells with lowered or absent MHC-I or induced MHC-I-related molecules while maintaining their tolerance toward self-MHC. Both CTL and mature NK cells undergo membranous reorganization and express various effector molecules to eliminate aberrant cells undergoing a stress of transformation, infection or other pathological noxa. Here, we present the cellular modules that underlie the CTL–NK circuitry to maximize their effector cooperativity against stressed or cancerous cells.

Contriving a chimeric polyvalent vaccine to prevent infections caused by herpes simplex virus (type-1 and type-2): an exploratory immunoinformatic approach

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) cause a variety of infections including oral-facial infections, genital herpes, herpes keratitis, cutaneous infection and so on. To date, FDA-approved licensed HSV vaccine is not available yet. Hence, the study was conducted to identify and characterize an effective epitope based polyvalent vaccine against both types of Herpes Simplex Virus. The selected proteins were retrieved from ViralZone and assessed to design highly antigenic epitopes by binding analyses of the peptides with MHC class-I and class-II molecules, antigenicity screening, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach. The final vaccine was constructed by the combination of top CTL, HTL and BCL epitopes from each protein along with suitable adjuvant and linkers. Physicochemical and secondary structure analysis, disulfide engineering, molecular dynamic simulation and codon adaptation were further employed to develop a unique multi-epitope peptide vaccine. Docking analysis of the refined vaccine structure with different MHC molecules and human immune TLR-2 receptor demonstrated higher interaction. Complexed structure of the modeled vaccine and TLR-2 showed minimal deformability at molecular level. Moreover, translational potency and microbial expression of the modeled vaccine was analyzed with pET28a(+) vector for E. coli strain K12 and the vaccine constructs had no similarity with entire human proteome. The study enabled design of a novel chimeric polyvalent vaccine to confer broad range immunity against both HSV serotypes. However, further wet lab based research using model animals are highly recommended to experimentally validate our findings. Communicated by Ramaswamy H. Sarma

Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the β2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.

Transcripts expressed in cytomegalovirus latency coding for an antigenic IE/E phase peptide that drives "memory inflation".

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Assembly, Intracellular Transport, and Release of MHC Class II Peptide Receptors.

MHC class II molecules play a pivotal role for the induction and maintenance of immune responses against pathogens, but are also implicated in pathological conditions like autoimmune diseases or rejection of transplanted organs. Human antigen-presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are, with the exception of DRα, composed of highly polymorphic α and β subunits. The combination of α- and β-chains results in a multitude of MHC-II αβ-heterodimers of the same isotype, but also isotype-mixed MHC class II molecules have been identified. Invariant chain chaperones the assembly of MHC-II molecules within the endoplasmatic reticulum and also facilitates the intracellular transport to MHC class II loading compartments (MIICs). MHC-II molecules are loaded with antigenic peptides and shuttled to the cell surface for inspection by CD4 T-cells. Alternatively, class-II molecules enriched on intraluminal vesicles can be released via exosomes into the extracellular space. Since some of the αβ-combinations may yield mismatched nonfunctional heterodimers, it is not entirely clear which type of HLA class II peptide receptors are transported to MIICs and found on the cell surface of antigen-presenting cells. We present techniques to inspect assembly, intracellular transport, cell surface expression, and exosomal release of MHC class II heterodimers.

Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing.

Killer cell immunoglobulin-like receptors (KIR), considered the most polymorphic natural killer (NK) cell regulators, bind HLA class-I molecules or still unknown ligands. Interest in KIR genotyping is increasing because of the importance of these receptors for identifying the best possible donor in hematopoietic stem cell transplantation to obtain a graft-versus-leukemia effect. Currently, routine protocols to determine the gene content of the KIR cluster are exclusively performed by PCR-SSO and PCR-SSP. To improve the study of these genes, we developed a multiplex, long-range PCR strategy suitable for simultaneous, high-resolution HLA class I and KIR genotyping by next generation sequencing (NGS). This protocol allows amplification of the 14 KIR genes, 2 KIR pseudogenes, and HLA class I genes, with subsequent sequencing on an Illumina platform. The bioinformatics analysis for KIR genotyping was performed by virtual hybridization of gene-specific probes, and HLA genotyping was done by GenDx NGSengine software. To validate the method reliability, 192 genomic DNA samples previously characterized by PCR-SSO were used. When a specific KIR gene was present, a large number of gene-specific virtual probes were detected, whereas when it was absent, very few or none were found, enabling cutoff establishment. Concordance for both the KIR and HLA assignments as compared with the previous characterization was 100%. In conclusion, the multiplex PCR NGS-based strategy presented could provide an efficient, less costly method for KIR-ligand genotyping by gene presence/absence. Furthermore, allele resolution will be possible when KIR-specific software becomes available.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition.

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host’s HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1–3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

1185 DNA hypermethylation of MHC class-I genes is associated with reduced expression and survival in melanoma

The aim of this study is to determine the role of DNA methylation in regulating expression of MHC class-I associated genes in melanoma. Reduced MHC class-I expression on the tumor cell surface is an important mechanism of escaping immunosurveillance, and is commonly seen in melanoma. DNA methylation is an epigenetic change that can silence gene expression, and has been implicated in regulating antigen expression and recognition in several cancer types. Abnormal DNA methylation is nearly universal in melanoma, however its role in regulating MHC molecule expression in melanoma is largely unknown. In this study, we investigated the methylation of MHC class-I associated molecules in melanoma tumors from The Cancer Genome Atlas. We uncovered that DNA hypermethylation of MHC class-I genes is a frequent event in melanoma, that hypermethylation is significantly associated with reduced gene expression, and identify the key CpG loci regulating expression. We found that methylation of beta-2 Microglobulin (B-2M) and HLA-B, in particular, may play important roles in melanoma survival. Patients with hypermethylation of B-2M exhibited a twofold decrease in median overall survival (HR =1.73, p=0.0001, Log-rank test). Hypermethylation of HLA-B was associated with significantly decreased 5-year overall survival (48% vs 68%, HR=1.68, p=0.0001, Log-rank test). Univariate and multivariate Cox proportional hazard analysis showed that DNA methylation of B-2M (p=5.8e-8) and HLA-B (p=5.15e-5) are prognostic factors of melanoma overall survival, independent of pathologic stage, age, gender, ulceration or Breslow thickness. Our results suggest that DNA hypermethylation may silence MHC class-I expression, and negatively impact melanoma survival. Based on our findings, targeting DNA methylation of B-2M and HLA-B may be a therapeutic strategy to restore expression of HLA class-I molecules, and potentially achieve improved immune responses in melanoma.

Effect of Polysorbate 80 and Particle Size of Budesonide API on In-vitro Dissolution Profiles of Budesonide MUPS Tablets 9 mg

The aim of this work was to study the effect of Polysorbate 80 concentration and particle size of Budesonide API on in-vitro dissolution profiles. Budesonide API was a BCS class-II molecule which is having low solubility and high permeability. To increase solubility particle size need to be reduced by wet milling technique using high-pressure homogenizer, Budesonide API is highly hydrophobic in nature, hence it was difficult to wet the API in purified water without wetting agent like Polysorbate-80. The optimal formulation was manufactured by using wet milling technique by using Polysorbate 80 as a wetting agent. The wet milled drug suspension was sprayed on sugar spheres, which was coated with Eudragit L100 and S100 to target the drug release in colon region, further coated with protective layer coating and compressed into MUPS tablets. Polysorbate 80 of 5 mg/unit with API particle size of 1.6 microns was found to be producing similar dissolution profile with the marketed formulation in all media from pH 4.5 to pH 7.5.

P120 Predicted indirectly recognizable HLA epitopes presented by HLA-DRB1 are related to HLA antibody formation during pregnancy

Aim Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child-specific HLA antibodies. We previously demonstrated that donor-specific HLA antibody formation after kidney transplantation is associated with donor-derived HLA epitopes presented by recipient HLA class-II (PIRCHE-II). In the present study we evaluated the role of PIRCHE-II in the child-specific HLA antibody formation during pregnancy. Methods A total of 229 mother–child pairs were HLA-typed. For all mismatched HLA class-I molecules of the child (inherited paternal antigens (IPA), we subsequently predicted the number of HLA epitopes that can be presented by maternal HLA class-II molecules. Child-specific antigens were classified as either immunogenic HLA or non-immunogenic HLA based upon the presence of specific antibodies and correlated to PIRCHE-II numbers. Results Immunogenic HLA contained higher numbers of PIRCHE-II than non-immunogenic HLA (median increases 1.3-fold). Moreover, the probability of IPA-specific antibody production during pregnancy increased with the number of PIRCHE-II. Conclusions In conclusion, our data suggest that the number of PIRCHE-II is related to the formation of child-specific HLA antibodies during pregnancy. The present confirmation of the role of PIRCHE-II in antibody formation outside the transplantation setting suggests that the PIRCHE-II concept is a universal principle.

In silico identification and characterization of common epitope-based peptide vaccine for Nipah and Hendra viruses.

To explore a common B- and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV). Membrane proteins F, G and M of HeV and NiV were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B- and T-cell epitopes that shared maximum identity with HeV and NiV were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope (VDPLRVQWRNNSVIS) showed at least 66% identity with all NiV and HeV G protein sequences, while the 15-mer M-epitope (GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all NiV and HeV M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II (MHC II) and class-I (MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against HeV and NiV. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction.

The mechanism of de novo expression of programmed cell death-ligand 1 in squamous cell carcinoma of the lung.

Immune checkpoint mechanisms such as the programmed cell death-ligand 1-programmed cell death 1 (PD‑L1-PD‑1) axis are utilized by tumor cells to evade the cytotoxicity of effector immune cells. However, environmental factors responsible for the expression of PD‑L1 on tumor cells remain to be fully elucidated. We hypothesized that an immunological interaction with tumor-infiltrating CD8+ lymphocytes (CD8+ TILs) would contribute to PD‑L1 expression in tumor cells. To verify this hypothesis, we examined the effect of interferon-γ (IFN-γ), a cytokine secreted by CD8+ TILs, on PD‑L1 expression in pulmonary squamous cell carcinomas invitro. We also evaluated the expression of PD‑L1 and major histocompatibility complex (MHC) classI molecules on tumor cells and CD8+ TILs in squamous cell carcinomas of the lung (n=77) by immunohistochemistry. IFN-γ upregulated PD‑L1 expression on pulmonary squamous carcinoma cells, and the reaction was reversible. In cases where which MHC classI molecule-positive tumor cells were dominant (n=72, 93.5%), cases in which PD‑L1-positive tumor cells were dominant (PD‑L1+ tumor cell‑dominant cases; n=45) were more frequently observed than PD‑L1-negative tumor cell‑dominant cases (n=27) (P=0.006). The number of CD8+ TILs was significantly higher in PD‑L1+ tumor cell‑dominant cases compared with PD‑L1- tumor cell‑dominant cases (P=0.005). These data suggest that the de novo expression of PD‑L1 on tumor cells is upregulated by IFN-γ secreted from CD8+ TILs upon recognition of the tumor cells with an MHC classI molecule.

A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Human leukocyte antigen-G overexpression predicts poor clinical outcomes in low-grade gliomas

Overexpression of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex class-I molecule associated with immunosuppression, has been reported in various human malignancies. In the present study, we examined the role of HLA-G in gliomas. Clinical characteristics, mRNA expression microarrays and follow-up data pertaining to 293 patients with histologically confirmed gliomas were analyzed. The expression levels of HLA-G were compared between different grades of gliomas and correlated with progression-free survival (PFS) and overall survival (OS) to evaluate its prognostic value. We found that HLA-G was overexpressed in gliomas as compared to that in normal brain tissue samples (−1.288±0.265). The highest expression levels were in glioblastomas (GBMs), anaplastic gliomas (AGs) and low-grade gliomas (LGGs), in that order (0.328±0.778, 0.176±0.881, −0.388±0.686, respectively). Significant inter-group differences were observed between low-grade and high-grade glioma tissues (p<0.001 and p<0.001, t-test, AGs and GBMs, respectively). More astrocytoma patients exhibited increased HLA-G expression as compared to other LGG patients (p=0.004, Chi-square test). Significant differences were observed with respect to PFS and OS (p=0.009 and 0.032, log-rank test, for PFS and OS, respectively) between the high- and low-expression subgroups in patients with LGGs. On Cox regression analysis, overexpression of HLA-G appeared to be an independent predictor of clinical outcomes (p=0.007 and 0.026, for PFS and OS, respectively). Our results suggest that HLA-G expression may serve as a potential biomarker for predicting aggressive tumor grades of gliomas and for histological subtype of LGGs. Elevated HLA-G expression could serve as an independent predictor of poor clinical outcomes in patients with low-grade gliomas.

NLRC5 elicits antitumor immunity by enhancing processing and presentation of tumor antigens to CD8+ T lymphocytes

ABSTRACTCancers can escape immunesurveillance by diminishing the expression of MHC class-I molecules (MHC-I) and components of the antigen-processing machinery (APM). Developing new approaches to reverse these defects could boost the efforts to restore antitumor immunity. Recent studies have shown that the expression of MHC-I and antigen-processing molecules is transcriptionally regulated by NOD-like receptor CARD domain containing 5 (NLRC5). To investigate whether NLRC5 could be used to improve tumor immunogenicity, we established stable lines of B16-F10 melanoma cells expressing NLRC5 (B16-5), the T cell co-stimulatory molecule CD80 (B16-CD80) or both (B16-5/80). Cells harboring NLRC5 constitutively expressed MHC-I and LMP2, LMP7 and TAP1 genes of the APM. The B16-5 cells efficiently presented the melanoma antigenic peptide gp10025–33 to Pmel-1 TCR transgenic CD8+ T cells and induced their proliferation. In the presence of CD80, B16-5 cells stimulated Pmel-1 cells even without the addition of gp100 peptide, indicating that NLRC5 facilitated the processing and presentation of endogenous tumor antigen. Upon subcutaneous implantation, B16-5 cells showed markedly reduced tumor growth in C57BL/6 hosts but not in immunodeficient hosts, indicating that the NLRC5-expressing tumor cells elicited antitumor immunity. Following intravenous injection, B16-5 and B16-5/80 cells formed fewer lung tumor foci compared to control cells. In mice depleted of CD8+ T cells, B16-5 cells formed large subcutaneous and lung tumors. Finally, immunization with irradiated B16-5 cells conferred protection against challenge by parental B16 cells. Collectively, our findings indicate that NLRC5 could be exploited to restore tumor immunogenicity and to stimulate protective antitumor immunity.

CD8(+) Tcells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) Tcells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) Tcells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) Tcells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) Tcells. Such mechanism may reduce the chances for pathogen immune evasion.