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Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP., (© 2021. The Author(s).)

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 Δ ptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo . We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8 + T-cell-mediated killing of antigen-loaded target cells in vivo . Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.

To protect the health of sows and gilts, significant investments are directed toward the development of vaccines against infectious agents that impact reproduction. We developed an intrauterine vaccine that can be delivered with semen during artificial insemination to induce mucosal immunity in the reproductive tract. An in vitro culture of uterine epithelial cells was used to select an adjuvant combination capable of recruiting antigen-presenting cells into the uterus. Adjuvant polyinosinic:polycytidylic acid (poly I:C), alone or in combination, induced expression of interferon gamma, tumor necrosis factor alpha, and select chemokines. A combination adjuvant consisting of poly I:C, host defense peptide and polyphosphazene (Triple Adjuvant; TriAdj), which previously was shown to induce robust mucosal and systemic humoral immunity when administered to the uterus in rabbits, was combined with boar semen to evaluate changes in localized gene expression and cellular recruitment, in vivo . Sows bred with semen plus TriAdj had decreased γδ T cells and monocytes in blood, however, no corresponding increase in the number of monocytes and macrophages was detected in the endometrium. Compared to sows bred with semen alone, sows bred with semen plus TriAdj showed increased CCL2 gene expression in the epithelial layer. These data suggest that the adjuvants may further augment a local immune response and, therefore, may be suitable for use in an intrauterine vaccine. When inactivated porcine parvovirus (PPV) formulated with the TriAdj was administered to the pig uterus during estrus along with semen, we observed induction of PPV antibodies in serum but only when the pigs were already primed with parenteral PPV vaccines. Recombinant protein vaccines and inactivated PPV vaccines administered to the pig uterus during breeding as a primary vaccine alone failed to induce significant humoral immunity. More trials need to be performed to clarify whether repeated intrauterine vaccination can trigger strong humoral immunity or whether the primary vaccine needs to be administered via a systemic route to promote a mucosal and systemic immune response., (Copyright © 2020 Hamonic, Pasternak, Ng, Fourie, Simko, Deluco and Wilson.)

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and tumors. Their TCRs respond to the sensing of isoprenoid metabolites, such as ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate, by butyrophilin (BTN) 3A1. BTN3A1 is an Ig superfamily protein with extracellular IgV/IgC domains and intracellular B30.2 domains that bind prenyl pyrophosphates. We have proposed that intracellular α helices form a coiled-coil dimer that functions as a spacer for the B30.2 domains. To test this, five pairs of anchor residues were mutated to glycine to destabilize the coiled-coil dimer. Despite maintaining surface expression, BTN3A1 mutagenesis either abrogated or decreased stimulation by ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. BTN3A2 and BTN3A3 proteins and orthologs in alpacas and dolphins are also predicted to have similar coiled-coil dimers. A second short coiled-coil region dimerizes the B30.2 domains. Molecular dynamics simulations predict that mutation of a conserved tryptophan residue in this region will destabilize the dimer, explaining the loss of stimulation by BTN3A1 proteins with this mutation. The juxtamembrane regions of other BTN/BTN-like proteins with B30.2 domains are similarly predicted to assume α helices, with many predicted to form coiled-coil dimers. An exon at the end of this region and the exon encoding the dimerization region for B30.2 domains are highly conserved. We propose that coiled-coil dimers function as rod-like helical molecular spacers to position B30.2 domains, as interaction sites for other proteins, and as dimerization regions to allow sensing by B30.2 domains. In these ways, the coiled-coil domains of BTN3A1 play critical roles for its function., (Copyright © 2019 by The American Association of Immunologists, Inc.)

SUM-IAP is an aldo-ifosfamide-perhydrothiazine derivative that, under physiological conditions, spontaneously hydrolyzes to SUM-aldo-ifosfamide. In SUM-IAP, one 2-chloroethyl group of the alkylating function of aldo-ifosfamide-perhydrothiazine is substituted by a mesyl-ethyl group. The compound was synthesized to investigate the influence of the alkylating function of aldo-ifosfamide on the antitumor activity of oxazaphosphorine cytostatics. In chemotherapy experiments in CD2F1 mice with advanced subcutaneously growing P388 mice leukemia cells, the primary tumor was reduced below the detection level with two highly dosed injections on days 7 and 8, but after 14 days, the primary tumor was measurable again. The primary tumor and detectable metastases killed the animals 29-30 days after SUM-IAP application. When, however, the animals were treated again with two highly dosed injections on day 14 and 15, a 4-5 times increase in the number of leukocytes was measured and all animals survived the observation period of 100 days. In the high, cancer-reductive dose range, SUM-IAP is not only a cytotoxic but also an immunostimulating oxazaphosphorine cytostatic.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O , O -diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β -d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)М^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.

The study aimed to identify an immunoregulatory factor that restores the phosphoantigen response of Vγ9Vδ2 + T cells from HIV-positive individuals on antiretroviral therapy. It was designed to characterize the effects of interleukin-18 (IL-18) on proliferation and effector function in Vγ9Vδ2 T cells from HIV-negative individuals and test whether exogenous IL-18 reconstitutes the Vγ9Vδ2 T-cell response to phosphoantigen from HIV-positive donors. Vγ9Vδ2 T cells from HIV-negative individuals responded strongly to phosphoantigen or aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMC), whereas cells with similar T-cell receptor profiles from HIV-positive individuals only responded to aminobisphosphonate. Interleukin-18 was higher after aminobisphosphonate stimulation due to activation of the inflammasome pathway. Both IL-18 and IL-18 receptor levels were measured and the activity of exogenous IL-18 on HIV-negative and HIV-positive PBMC was evaluated in terms of Vγ9Vδ2 T-cell proliferation, memory subsets, cytokine expression and CD107a expression. Interleukin-18 stimulation increased proliferation, enhanced the accumulation of effector memory cells, and increased expression of cytotoxic markers in HIV-negative controls. When Vγ9Vδ2 T cells from HIV-positive individuals were stimulated with isopentenyl pyrophosphate in the presence of IL-18, there was increased proliferation, accumulation of memory cells, and higher expression of CD56, NKG2D and CD107a (markers of cytotoxic effector phenotype). Interleukin-18 stimulation specifically expanded the Vγ9-JγP + subset of Vγ9Vδ2 T cells, as was expected for normal responses to phosphoantigen. Interleukin-18 is a potent stimulator of Vγ9Vδ2 T-cell proliferation and effector function. Therapies directed at reconstituting Vγ9Vδ2 T-cell activity in HIV-positive individuals should include stimulators of IL-18 or direct cytokine supplementation., (© 2017 John Wiley & Sons Ltd.)

Two macromolecular immunoadjuvants, poly[di(carboxylatophenoxy)phosphazene], PCPP, and poly[di(carboxylatoethylphenoxy)phosphazene], PCEP, have been investigated for their molecular interactions with model and biopharmaceutically important proteins in solutions, as well as for their TLR stimulatory effects and pH-dependent membrane disruptive activity in cellular assays. Solution interactions between polyphosphazenes and proteins, including antigens and soluble immune receptor proteins, have been studied using Asymmetric Flow Field Flow Fractionation (AF4) and Dynamic Light Scattering (DLS) at near physiological conditions: phosphate buffered saline, pH 7.4. Polyphosphazenes demonstrated selectivity in their molecular interactions with various proteins, but displayed strong binding with all vaccine antigens tested in the present study. It was found that both PCPP and PCEP showed strong avidity to soluble immune receptor proteins, such as Mannose Receptor (MR) and certain Toll-Like Receptor (TLR) proteins. Studies on TLR stimulation in vitro using HEK293 cells with overexpressed human TLRs revealed activation of TLR7, TLR8, and TLR9 signaling pathways, albeit with some nonspecific stimulation, for PCPP and the same pathways plus TLR3 for PCEP. Finally, PCEP, but not PCPP, demonstrated pH-dependent membrane disruptive activity in the pH range corresponding to the pH environment of early endosomes, which may play a role in a cross-presentation of antigenic proteins.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and young children. There are no licensed RSV vaccines available, and the few treatment options for high-risk individuals are either extremely costly or cause severe side effects and toxicity. Immunomodulation mediated by a novel formulation consisting of the toll-like receptor 3 agonist poly(I:C), an innate defense regulator peptide and a polyphosphazene (P-I-P) was evaluated in the context of lethal infection with pneumonia virus of mice (PVM). Intranasal delivery of a single dose of P-I-P protected adult mice against PVM when given 24 h prior to challenge. These animals experienced minimal weight loss, no clinical disease, 100% survival, and reduced lung pathology. Similar clinical outcomes were observed in mice treated up to 3 days prior to infection. P-I-P pre-treatment induced early mRNA and protein expression of key chemokine and cytokine genes, reduced the recruitment of neutrophils and eosinophils, decreased virus titers in the lungs, and modulated the delayed exacerbated nature of PVM disease without any short-term side effects. On day 14 post-infection, P-I-P-treated mice were confirmed to be PVM-free. These results demonstrate the capacity of this formulation to prevent PVM and possibly other viral respiratory infections., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies., (Copyright © 2016 by The American Association of Immunologists, Inc.)

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB., (Copyright © 2016 Xia et al.)

Protein phosphorylation is one of the most common and extensively studied post-translational modifications (PTMs). Compared to the O-phosphorylation on Ser, Thr and Tyr residues, our understanding of arginine phosphorylation is relatively limited, both in prokaryotes and eukaryotes, due to the intrinsic instability of phosphoarginine (pArg) and the lack of a feasible method to produce anti-pArg antibodies. We report the design and synthesis of a stable pArg analog, in which the labile N-P bond is replaced with a non-hydrolyzable C-P bond. Significantly, this analog was successfully used as a hapten to raise an immune response and the first mouse polyclonal antibody that specifically recognizes pArg-containing peptides and proteins was produced using analog-KLH conjugated as the immunogen. The generated antibody shows excellent specificity towards pArg-containing peptides and proteins, and could be used for a variety of biological detection methods. This provides us an invaluable tool to unravel the mystery of the biological function of pArg.

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

γδ T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human γδ T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the γδ T cell compartment in cancer immunosurveillance and paving the way for the use of γδ TCRs in cancer therapy. Ligand recognition by the γδ TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike αβ T cells, γδ T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human γδ T cells and discuss the promise of harnessing these cells for cancer treatment.

Experiments on noninbred albino rats showed that a chronic exposure to organophosphorus compounds (carbophos and metaphos, 30 days, total dose 0.3 LD50) is primarily followed by a decrease in the immune reactions and IFN-γ associated with Th1 lymphocyte function (in comparison with the immune response due to activation of Th2 cells by IL-4). The concentrations of IL-2, IL-6, and IL-10 in the blood decreased after 30-day intoxication. The immune reactions associated with functional activity of Th1 and Th2 lymphocytes were shown to decrease similarly after chronic treatment with organophosphorus compounds for 60 days (total dose 0.6 LD50). This exposure was accompanied by a decrease in the concentrations of IFN-γ, IL-4, IL-2, and IL-6, but had no effect on the level of IL-10 in the blood.

Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 mAb stimulation because IL-2 is released by β(-) Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophosphates but does not bind the intermediates with high affinity.

Exposing human tumor cells to nitrogen-containing bisphosphonates, such as zoledronic acid (Zol), greatly increases their susceptibility to killing by γδ T cells. Based on this finding and other studies, cancer immunotherapy using γδ T cells and nitrogen-containing bisphosphonates has been studied in pilot clinical trials and has shown benefits. Although Zol treatment can render a wide variety of human tumor cells susceptible to γδ T cell killing, there has not been a systematic investigation to determine which types of tumor cells are the most susceptible to γδ T cell-mediated cytotoxicity. In this study, we determined the Zol concentrations required to stimulate half maximal tumor necrosis factor-α production by γδ T cells cultured with various tumor cell lines pretreated with Zol and compared these concentrations with those required for half maximal inhibition of farnesyl diphosphate synthase (FPPS) in the same tumor cell lines. The inhibition of tumor cell growth by Zol was also assessed. We found that FPPS inhibition strongly correlated with γδ T cell activation, confirming that the mechanism underlying γδ T cell activation by Zol is isopentenyl diphosphate (IPP) accumulation due to FPPS blockade. In addition, we showed that γδ T-cell receptor-mediated signaling correlated with γδ T cell tumor necrosis factor-α production and cytotoxicity. Some lymphoma, myeloid leukemia, and mammary carcinoma cell lines were relatively resistant to Zol treatment, suggesting that assessing tumor sensitivity to Zol may help select those patients most likely to benefit from immunotherapy with γδ T cells., (© 2013 Japanese Cancer Association.)

Among the strategies aimed at biocompatible means for organophosphorus nerve agents neutralization, immunoglobulins have attracted attention in the 1990's and 2000's both for their ability to immobilize the toxicants, but also for their ability to be turned into enzymatically active antibodies known as catalytic antibodies or abzymes (antibodies--enzymes). We will present here a critical review of the successive strategies used for the selection of these nerve agent-hydrolyzing abzymes, based on hapten design, namely antibodies raised against a wide variety of transition state analogs, and eventually the strategies based on anti-idiotypic antibodies and reactibodies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Nitrogen-containing bisphosphonates (N-BP) such as zoledronate and risedronate exhibit antitumor effects. They block the activity of farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway, leading to intracellular accumulation of mevalonate metabolites (IPP/ApppI), which are recognized as tumor phosphoantigens by Vγ9Vδ2 T cells. However, mechanisms responsible for Vγ9Vδ2 T-cell recognition of N-BP-treated tumors producing IPP/ApppI remain unclear., Experimental Design: The effects of N-BPs on Vγ9Vδ2 T-cell expansion and anticancer activity were evaluated in vitro and in animal models of human breast cancers. The modalities of recognition of breast tumors by Vγ9Vδ2 T cells in N-BP-treated animals were also examined., Results: We found a strong correlation between Vγ9Vδ2 T-cell anticancer activity and intracellular accumulation of IPP/ApppI in risedronate-treated breast cancer cells in vitro. In addition, following risedronate treatment of immunodeficient mice bearing human breast tumors, human Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels but not those expressing low IPP/ApppI levels. The combination of doxorubicin with a N-BP improved, however, Vγ9Vδ2 T-cell cytotoxicity against breast tumors expressing low IPP/ApppI levels. Moreover, Vγ9Vδ2 T-cell cytotoxicity in mice treated with risedronate or zoledronate did not only depend on IPP/ApppI accumulation in tumors but also on expression of tumor cell surface receptor intercellular adhesion molecule-1 (ICAM-1), which triggered the recognition of N-BP-treated breast cancer cells by Vγ9Vδ2 T cells in vivo., Conclusion: These findings suggest that N-BPs can have an adjuvant role in cancer therapy by activating Vγ9Vδ2 T-cell cytotoxicity in patients with breast cancer that produces high IPP/ApppI levels after N-BP treatment., (©2012 AACR.)

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis is an autoimmune disease that primarily affects the limbs, but the pathogenic mechanism remains unclear. γδ T cells, a T-cell subpopulation, are characterized by multiple biological functions and associated with a variety of diseases. This study investigated the antigen-presenting effects of γδ T cells and their relationship with rheumatoid arthritis development. We found that Vγ9Vδ2 T cells (the predominant subtype of γδ T cells in peripheral blood) were activated by isopentenyl pyrophosphate to continuously proliferate and differentiate into effector memory cells. The effector memory Vγ9Vδ2 T cells exhibited phenotypic characteristics of specific antigen-presenting cells, including high HLA-DR and CD80/86 expression. These Vγ9Vδ2 T cells could present soluble antigens and synthetic peptides to CD4(+) T cells. Vγ9Vδ2 T cells with different phenotypes showed different cytokine secretion patterns. Effector memory Vγ9Vδ2 T cells simultaneously secreted not only interferon (IFN)-γ but also IL-17. The peripheral blood and joint synovial fluid from RA patients contained numerous heterogeneous γδ T cells that were predominantly effector memory Vγ9Vδ2 T cells with the ability to secrete inflammatory factors. We also found that γδ T cells had a similar antigen-presenting capability to B cells. These results suggest that during the development of rheumatoid arthritis, γδ T cells can aggravate immune dysfunction and produce abnormal immune damage by secreting cytokines and inducing inflammatory cells to participate in synergistic inflammatory responses. Furthermore, γδ T cells can behave similarly to B cells to present viral peptides and autoantigen peptides to CD4(+) T cells, thus sustaining CD4(+) T-cell activation.

The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9(+)Vδ2(+) (Vγ9(+)) T cells, in JIA. We found that as opposed to CD4(+) T cells, equally high percentages (∼35%) of Vγ9(+) T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2(+) T cells, similarly amplified cytokine secretion by SF and PB Vγ9(+) T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9(+) T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9(+) T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4(+)CD25(+)FOXP3(+) cells (regulatory T cells). Furthermore, coculture with the Vγ9(+) T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9(+) T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.

A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL(-1). The time required for the equilibrium of antibody-antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Class II major histocompatibility molecules are the primary susceptibility locus for many autoimmune disorders, including type 1 diabetes. Human DQ8 and I-A(g7), in the NOD mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We used an in silico molecular docking program to screen a large "druglike" chemical library to define small molecules capable of occupying specific structural pockets along the I-A(g7) binding groove, with the objective of influencing presentation to T cells of the autoantigen insulin B chain peptide consisting of amino acids 9-23. In this study we show, using both murine and human cells, that small molecules can enhance or inhibit specific TCR signaling in the presence of cognate target peptides, based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent, with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing responses to peptide. At nanomolar concentrations, the inhibitory molecules block the insulin B chain peptide consisting of amino acids 9-23, endogenous insulin, and islet-stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses, with potentially therapeutic applications.

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8(+) αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8(+) αβ antitumor effector T cells.

Human Vγ2 Vδ2-bearing T cells have recently received much attention in cancer immunotherapy. In this study, we conducted a phase I/II clinical trial of the adoptive transfer of γδ T cells to patients with advanced renal cell carcinoma. Eleven patients who had undergone nephrectomy and had lung metastasis were enrolled. Peripheral blood γδ T cells obtained from the patients were stimulated ex vivo with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), a synthetic pyrophosphomonoester antigen, and transferred in combination with zoledronic acid (Zol) and teceleukin (recombinant human interleukin-2). Expanded γδ T cells exhibited potent cytotoxic activity against tumor cells in vitro, and the proportion of peripheral blood γδ T cells among CD3(+) cells typically peaked three to 5 days after transfer. Tumor doubling time was prolonged in all 11 patients, and the best overall responses were 1 CR, 5 SD, and 5 PD, as defined based on Response Evaluation Criteria in Solid Tumors (RECIST). Although ten patients developed adverse reactions of grade ≥3, they were likely to have been the result of the concomitant infusion of Zol and IL-2, and most symptoms swiftly reverted to normal during the course of treatment. In conclusion, this clinical trial demonstrated that our regimen for the adoptive transfer of γδ T cells in combination with Zol and IL-2 was well tolerated and that objective clinical responses could be achieved in some patients with advanced renal cell carcinoma.

Purpose of Review: Several clinical and epidemiological studies have reported an association between exposure to pesticides, bronchial hyper-reactivity and asthma symptoms. This article reviews the mechanistic evidence lending support to the concept that either acute or chronic low-level inhalation of pesticides may trigger asthma attacks, exacerbate asthma or increase the risk of developing asthma., Recent Findings: Pesticide aerosols or gases, like other respiratory irritants, can lead to asthma through interaction with functional irritant receptors in the airway and promoting neurogenic inflammation. Cross-talk between airway nerves and inflammatory cells helps to maintain chronic inflammation that eventually damages the bronchial epithelium. Certain organophosphorus insecticides cause airway hyper-reactivity via a common mechanism of disrupting negative feedback control of cholinergic regulation in the lungs. These pesticides may interact synergistically with allergen sensitization rendering individuals more susceptible for developing asthma., Summary: Many pesticides are sensitizers or irritants capable of directly damaging the bronchial mucosa, thus making the airway very sensitive to allergens or other stimuli. However, most pesticides are weakly immunogenic so that their potential to sensitize airways in exposed populations is limited. Pesticides may increase the risk of developing asthma, exacerbate a previous asthmatic condition or even trigger asthma attacks by increasing bronchial hyper-responsiveness.

Programmed cell death-1 (PD-1) is an inhibitory receptor and plays an important role in the regulation of αβ T cells. Little is known, however, about the role of PD-1 in γδ T cells. In this study, we investigated the expression and function of PD-1 in human γδ T cells. Expression of PD-1 was rapidly induced in primary γδ T cells following antigenic stimulation, and the PD-1(+) γδ T cells produced IL-2. When PD-1(+) γδ T cells were stimulated with Daudi cells with and without programmed cell death ligand-1 (PD-L1) expression, the levels of IFN-γ production and cytotoxicity in response to PD-L1(+) Daudi cells were diminished compared to the levels seen in response to PD-L1(-) Daudi cells. The attenuated effector functions were reversed by anti-PD-L1 mAb. When PD-1(+) γδ T cells were challenged by PD-L1(+) tumors pretreated with zoledronate (Zol), which induced γδ TCR-mediated signaling, the resulting reduction in cytokine production was only slight to moderate compared to the reduction seen when PD-1(+) γδ T cells were challenged by PD-L1(-) tumors. In addition, cytotoxic activity of PD-1(+) γδ T cells against Zol-treated PD-L1(+) tumors was comparable to that against Zol-treated PD-L1(-) tumors. These results suggest that TCR triggering may partially overcome the inhibitory effect of PD-1 in γδ T cells., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human Vγ9Vδ2 T cells are potent anti-tumor lymphocytes that specifically respond to pyrophosphate (phospho-) antigens, which constitute the basis of current γδ T-cell-based immunotherapy strategies. Despite a clear involvement of the TCR, the costimulation requirements of Vγ9Vδ2 T cells remain ill-defined. Here, we show that the expression of the CD27 receptor by the vast majority of Vγ9Vδ2 peripheral blood lymphocytes endows them with enhanced proliferative capacity upon ligation by its unique ligand CD70, a tumor necrosis factor superfamily member expressed on lymphoma B-cells but also on TCR-activated γδ T cells. Moreover, Vγ9Vδ2 T-cell treatment with soluble recombinant CD70 induced calcium signals and increased transcription of anti-apoptotic Bcl2a1 and cell-cycle-promoting Cyclin D2 genes. We further demonstrate that the manipulation of CD70-CD27 interactions significantly impacted on Vγ9Vδ2 T-cell survival, proliferation and cytokine secretion, in both loss-of-function and gain-of-function experiments. Thus, CD27 coreceptor signals strongly promoted the expansion of Th1-biased, CD27(+) Vγ9Vδ2 peripheral blood lymphocytes in the context of TCR-mediated stimulation with phosphoantigens. These data collectively establish a novel role for the CD70-CD27 axis in human γδ T-cell activation and hence open new perspectives for its modulation in clinical settings., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Embolization with microspheres is widely applied to treat uterine fibroids. However, the foreign body reaction that could result from the degradation of the microspheres remains to be evaluated to adequately appreciate the tissular tolerance to such biomaterials. We compared herein the in situ degradation of PMMA microspheres coated with polyphosphazene (PMMA-PPms) and trisacryl gelatin microspheres (TGms) and we thoroughly investigated the induced local inflammatory responses, at 1 and 4 weeks after uterine artery embolization in sheep, by using immunohistochemistry and microarray analyses. PMMA-PPms underwent an acute and partial degradation that was associated with the early recruitment of phagocytic cells (CD172a+ and MHCII+), and with the up-regulated expression of genes involved in the movement of phagocytes (ALOX5AP, CXCL2, CXCL5, IL8, PTGS2, YARS). By contrast, TGms were not degraded and triggered a different inflammation profile including the recruitment of FBR Giant Cells and T-lymphocytes (CD4+) and the increased expression of genes involved in lymphocyte activation (CXCL10, IL2RG, IRAK4, MALT1). Our results indicate that, in contrast to a non-degradable microsphere such as TGms which is associated to a poorly inflammatory foreign body reaction that rapidly resolves, PMMA-PPms, which is partially degradable, rapidly recruits and activates inflammatory phagocytes, thus delaying the resolution of the foreign body reaction., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.

A broad-selective monoclonal antibody (Mab) for organophosphorus (OP) pesticides was raised using heterologous indirect enzyme-linked immunosorbent assay (ELISA) to screen hybridomas. On the basis of this Mab, five coating antigens were used to develop homologous and heterologous indirect competitive ELISAs. With the most suitable competitor, a sensitive and broad-selective ELISA was developed. The IC(50) values were estimated to be 20.32 ng/mL for parathion, 21.44 ng/mL for methyl-parathion, 42.15 ng/mL for fenitrothion, and 58.85 ng/mL for isocarbophos. Spike recoveries were between 70.52 and 103.27% for the detection of single pesticide residues of the four OP pesticides in purple-clayed paddy soil. Moreover, the chosen ELISA was then applied to the detection of mixtures of parathion and methyl-parathion in soil samples. The average recovery and coefficient of variation were 80.91 and 4.82%, respectively. Results proved that this broad-selective ELISA would be useful for the multiresidue determination of OP pesticides.

CRX-527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates, which are considered as potential vaccine adjuvants or stand-alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane-bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527. First, we found that CRX-527 induces NF-kappaB and interferon regulatory factor-3 (IRF-3) activation in human embryonic kidney cells transfected with TLR4 and MD-2 genes alone, whereas the responses to LPS require either co-transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte-derived DC, which are devoid of mCD14 respond to CRX-527 but not to LPS in serum-free medium. Furthermore, we found that, in contrast to LPS, CRX-527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14-dependent responses are defective. Finally, we demonstrated that splenocytes from CD14-deficient mice produce cytokines in response to CRX-527 but not to LPS. We conclude that the lipid A mimetic CRX-527 does not require the CD14 co-receptor to elicit TLR4-mediated responses.

Human Vγ9Vδ2 T cells recognise pyrophosphate-based antigens (phosphoantigens) and have multiple functions in innate and adaptive immunity, including a unique ability to activate other cells of the immune system. We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP) in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under these conditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24-72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.

Intradermal immunization using microfabricated needles represents a potentially powerful technology, which can enhance immune responses and provide antigen sparing. Solid vaccine formulations, which can be coated onto microneedle patches suitable for simple administration, can also potentially offer improved shelf-life. However the approach is not fully compatible with many vaccine adjuvants including alum, the most common adjuvant used in the vaccine market globally. Here, we introduce a polyphosphazene immuno adjuvant as a biologically potent and synergistic constituent of microneedle-based intradermal immunization technology. Poly[di(carboxylatophenoxy)phosphazene], PCPP, functions both as a vaccine adjuvant and as a key microfabrication material. When used as part of an intradermal delivery system for hepatitis B surface antigen, PCPP demonstrates superior activity in pigs compared to intramascular administration and significant antigen sparing potential. It also accelerates the microneedle fabrication process and reduces its dependence on the use of surfactants. In this way, PCPP-coated microneedles may enable effective intradermal vaccination from an adjuvanted patch delivery system.

Objective: To develop an immunoassay for multiple residues of organophosphorus pesticides., Methods: The generic hapten O, O-dimethyl-O-(4-carboxyphenyl)ester (HP) was synthesized by the reaction between O, O-Dimethyl phosphorochloridothionate and 4-hydroxybenzoic acid methyl ester, and then conjugated to bovine serum albumin (BSA) by active ester method for immunogens and ovalbumin (OVA) by mixed anhydride reaction for coating antigens., Results: The hapten was synthesized successfully, which was characterized by MS. The coupling ratios were 15.6:1 (HP-BSA) and 16.2:1(HP-OVA), respectively, calculated by UV spectrophotometry., Conclusion: suggested that the artificial antigens could be used for production of broad specificity anti-organophosphorous pesticides antibody.

Objective: To evaluate Vgamma2Vdelta2 T cells in a group of HIV-infected patients who suppress HIV replication without antiretroviral therapy (natural viral suppressors, NVSs)., Design: : It is a cross-sectional study., Methods: We compared Vgamma2Vdelta2 T-cell frequency, T-cell repertoire, and responses to isopentenyl pyrophosphate stimulation between NVSs (n = 21) and HIV-uninfected controls (n = 27) and between NVSs and HIV-infected patients taking HAART with suppressed viral replication (HIV-P; n = 25)., Results: NVSs had a mean frequency of 1.06 +/- 0.82% CD3Vdelta2+ cells among total lymphocytes, which was significantly higher than both control groups (HIV-negative: 0.50 +/- 0.53%, P = 0.042; HIV-P: 0.34 +/- 0.37%, P = 0.002). The proportion of Vgamma2 chains correlating with the Vgamma2-Jgamma1.2 rearrangement was reduced among NVSs compared with HIV-negative controls (0.57 +/- 0.06 vs. 0.32 +/- 0.04; P = 0.016) but was increased compared with HIV-P patients (0.32 +/- 0.04 vs. 0.22 +/- 0.03; P = 0.03). NVSs had a similar baseline frequency of CD27/CD45RA effector cells (19.6 +/- 4.2%) compared with HIV-negative controls (20.8 +/- 12.9%; P = 0.35)., Conclusion: The altered gammadelta T-cell receptor repertoire among NVS was consistent with the known effect of HIV-1 on these cells. Uniquely among all HIV-infected groups, NVS reconstituted the gammadelta T-cell population, eventually reaching levels significantly above controls. This capacity to recover gammadelta T-cell numbers and function distinguishes individuals who control HIV-1 with and without HAART.

Human Vgamma9Vdelta2 T lymphocytes recognize phosphorylated alkyl Ags. Isopentenyl pyrophosphate (IPP) was previously proposed as the main Ag responsible for Vgamma9Vdelta2 T cell activation by cancer cells. However, triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (ApppI), a metabolite in which the isopentenyl moiety is linked to ATP, was reported in cells activated with aminobisphosphonates. The contribution of this compound to tumor-stimulatory activity was thus examined. ApppI induces selective expansion of Vgamma9Vdelta2 T cells from PBMCs. In the absence of APCs, however, ApppI has little stimulatory activity on Vgamma9Vdelta2 T cells, and optimal activation with ApppI requires addition of a nucleotide pyrophosphatase releasing IPP plus AMP. Thus, ApppI has no intrinsic stimulatory activity. Nevertheless, stimulation by ApppI is strengthened by the presence of APCs. Moreover, in contrast to IPP, ApppI can be efficiently pulsed on dendritic cells as well as on nonprofessional APCs. Pulsed APCs display stable and phosphatase-resistant stimulatory activity, indicative of Ag modification. HPLC analysis of tumor cell extracts indicates that latent phosphoantigenic activity is stored intracellularly in the Vgamma9Vdelta2 cell-sensitive tumor Daudi and can be activated by a nucleotide pyrophosphatase activity. The presence of ApppI in Daudi cell extracts was demonstrated by mass spectrometry. Nucleotidic Ags such as ApppI are thus a storage form of phosphoantigen which may represent a major source of phosphoantigenic activity in tumor cells. The unique properties of ApppI may be important for the design of Ags used in anticancer immunotherapeutic protocols using Vgamma9Vdelta2 cells.

Lower numbers of Vgamma9Vdelta2 T cells in cord blood (CB) than in adult peripheral blood (PB), as well as their impaired ability to produce interferon-gamma (IFN-gamma) in response to stimulation, are associated with functional deficiency in the immune system in newborns. In this study, we stimulated CB Vgamma9 T cells with their T-cell receptor-specific ligand, isopentenyl pyrophosphate (IPP), plus exogenous costimulatory cytokines such as interleukin-2 (IL-2), IL-12 and tumour necrosis factor-alpha (TNF-alpha), which are known to play important roles in the activation of PB gammadelta T cells. Our data show that CB Vgamma9 T cells are able to produce IFN-gamma at levels comparable to PB Vgamma9 T cells by the addition of TNF-alpha in the presence of IPP and IL-2; however, under the same culture conditions, IL-12 does not efficiently activate CB Vgamma9 T cells to produce IFN-gamma. The frequency of TNF-alpha receptor II-positive Vgamma9T cells and the expression levels of TNF-alpha receptor II are similar in CB and PB; in contrast, the frequency of IL-12 receptor betaI (IL-12RbetaI) -positive Vgamma9T cells and expression levels of IL-12RbetaI are significantly lower in CB than PB. TNF-alpha but not IL-12 increases the expression of IL-2Rbeta on CB Vgamma9 T cells. These results provide new insights into the role of TNF-alpha in the activation of CB Vgamma9 T cells.

Soman is an organophosphorus neurotoxin which inhibits the activity of acetylcholinesterase (AChE). The goal of this work was to find out whether antibodies against an organophosphorus hapten could protect mice from soman toxicity. An organophosphorus hapten P6 was synthesized. Its chemical conjugates with limulus polyphemus hemocyanin and bovine serum albumin were used as immune antigen (P6-LPH) and detection antigen (P6-BSA), respectively. Eight hybridoma cell lines secreting monoclonal antibodies (Mabs) were established. The binding reactivities of Mabs with P6 and soman were determined by competitive inhibition enzyme immunoassay (CIEIA). All antibodies recognized P6 and four of them (2C10, 3G1, 3B9 and 3C11) combined with soman. The IC(50) was 10(-6.5) to 10(-5.3)mol/l for P6 and 10(-5) to 10(-3.5)mol/l for soman. Furthermore, Mab 3G1 reduced the inhibition of AChE activity by soman in vitro. When soman was pre-incubated with Mabs before being injected into mice, soman potency was reduced, indicating that Mabs could protect mice from soman toxicity. In an active immunization regimen, mice immunized with P6-LPH and challenged with 0.15mg/kg soman injected subcutaneously, had fewer signs of intoxication and a higher survival rate compared with control mice. These results demonstrate that the anti-soman antibodies have proper characteristics as scavengers in the detoxication of soman poisoning.

Vaccines consisting of subunit or protein antigens are less immunogenic than traditional vaccines, and therefore require formulation with an adjuvant. Conventional adjuvants, however, often cause undesirable injection site reactions and Th2-biased immune responses. Therefore, novel vaccine adjuvants which can safely enhance and selectively bias the resulting immune response are required. Here the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG+indol+PP) was evaluated for its ability to enhance and modulate the immune response when formulated with the antigen hen egg lysozyme (HEL). Cattle immunized with HEL co-adjuvanted with CpG+indol+PP developed higher antigen-specific humoral responses, and long-lasting cell-mediated immune responses, as evidenced by elevated levels of IFN-gamma secretion by re-stimulated PBMCs, that were superior even to EMULSIGEN((R)), an oil-in-water based adjuvant that was used as positive control. Physical characterization of the vaccines indicated that formulation of HEL with CpG+indol+PP resulted in the formation of antigen-adjuvant complexes, which may have contributed to their enhanced immunogenicity. Furthermore, the addition of polyphosphazene to CpG ODN and indolicidin dose-dependently enhanced the secretion of the cytokines IFN-alpha, TNF-alpha and IFN-gammain vitro, indicating that polyphosphazene can also synergize with CpG ODN and indolicidin to stimulate innate immune responses.

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-alpha, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-gamma secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.

Adjuvants are important components of vaccine formulations. Their functions include the delivery of antigen, recruitment of specific immune cells to the site of immunization, activation of these cells to create an inflammatory microenvironment, and maturation of antigen-presenting cells for enhancement of antigen-uptake and -presentation in secondary lymphoid tissues. Adjuvants include a large family of molecules and substances, many of which were developed empirically and without knowledge of their specific mechanisms of action. The discovery of pattern recognition receptors including Toll-like-, nucleotide-binding oligomerization domain (NOD)- and mannose-receptors, has significantly advanced the field of adjuvant research. It is now clear that effective adjuvants link innate and adaptive immunity by signaling through a combination of pathogen recognition receptors (PRRs). Research in our lab is focused towards the development of novel adjuvants and immunomodulators that can be used to improve neonatal vaccines for humans and animals. Using a neonatal pig model for pertussis, we are currently analyzing the effectiveness of host defence peptides (HDPs), bacterial DNA and polyphosphazenes as vaccine adjuvants.

Competitive enzyme-linked immunosorbent assays (ELISAs) in indirect and direct format were developed for the quantitative detection of the organophosphorus insecticide EPN. Five EPN derivatives (haptens) were synthesized and coupled to carrier proteins to use as an immunogen or as a competitor. Rabbits were immunized with two of the five haptens coupled to KLH for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin (OVA). Using the serum with the highest specificity and a coating antigen (hapten-OVA conjugate), an indirect (antigen-coated) ELISA was developed, which showed an IC(50) of 5.6 ng/mL with a detection limit of 0.2 ng/mL (20% inhibition). A direct (antibody-coated) ELISA using an enzyme tracer (hapten-enzyme conjugate) was also developed, which showed an IC(50) of 8.4 ng/mL with a detection limit of 0.9 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticide parathion-ethyl only in the direct ELISA. The recoveries of EPN from spiked samples determined by the indirect ELISAs were between 37 and 164%.

Vgamma2Vdelta2 T cells comprise the major subset of peripheral blood gammadelta T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the Vgamma2Vdelta2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the Vgamma2Vdelta2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C(5)-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to gammadelta T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, beta(2)-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C(5)-OPP. Finally, pulsing of BZ-(C)-C(5)-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.