Listeria monocytogenes-infected human monocytic derived dendritic cells activate Vγ9Vδ2 T cells independently of HMBPP production.

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.

Critical Roles for Coiled-Coil Dimers of Butyrophilin 3A1 in the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T Cells.

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.

Selective Destruction of Interleukin 23-Induced Expansion of a Major Antigen-Specific γδ T-Cell Subset in Patients With Tuberculosis.

A loss of antigen-specific T-cell responses due to defective cytokine signaling during infections has not been reported. We hypothesize that tuberculosis can destroy signaling effects of selective cytokine(s) and induce exhaustion of antigen-specific T cells. To test this hypothesis, mechanistic studies were performed to examine whether and how tuberculosis blocked interleukin 23 (IL-23) and interleukin 2 (IL-2) signaling effects on a major human γδ T-cell subpopulation, phosphoantigen HMBPP-specific Vγ2Vδ2 T cells. IL-23 and IL-2 significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection, and IL-2 synergized the effect of IL-23. IL-23-induced expansion of Vγ2Vδ2 T cells involved STAT3. Surprisingly, patients with tuberculosis exhibited a selective destruction of IL-23-induced expansion of these cells. The tuberculosis-driven destruction of IL-23 signaling coincided with decreases of expression and phosphorylation of STAT3. Interestingly, impairing of STAT3 was linked to marked increases in the microRNAs (miRNAs) hsa-miR-337-3p and hsa-miR-125b-5p in Vγ2Vδ2 T cells from patients with tuberculosis. Downregulation of hsa-miR-337-3p and hsa-miR-125b-5p by miRNA sponges improved IL-23-mediated expansion of Vγ2Vδ2 T cells and restored the ability of these cells to produce anti-tuberculosis cytokines. These results support our hypothesis that tuberculosis can selectively impair a cytokine effect while sparing another and can induce exhaustion of T cells in response to the respective cytokine.

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

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.

HMBPP Analog Prodrugs Bypass Energy-Dependent Uptake To Promote Efficient BTN3A1-Mediated Malignant Cell Lysis by Vγ9Vδ2 T Lymphocyte Effectors.

Vγ9Vδ2 effector T cells lyse cells in response to phosphorus-containing small molecules, providing primates a unique route to remove infected or malignant cells. Yet, the triggering mechanisms remain ill defined. We examined lysis mediated by human Vγ9Vδ2 effector T cells in response to the naturally occurring (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) or a synthetic cell-permeable prodrug, bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. CD27(+)/CD45RA(-) Th1-like effector cells killed K562 target cells through a mechanism that could be enhanced by either compound or TCR Ab and blocked by Src inhibition or butyrophilin 3 isoform A1 (BTN3A1) disruption. Pretreatment at 4 °: C decreased HMBPP-induced lysis but did not reduce lysis induced by bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate. Together, our results show that internalization of HMBPP into target cells is required for BTN3A1-dependent lysis by Vγ9Vδ2 effector T cells. The enhanced activity of the prodrug analog is due to its ability to bypass the pathways required for entry of HMBPP. These findings support an inside-out model of T cell triggering driven by small-molecule induction of BTN3A1.

Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2 T cells after Mycobacterium tuberculosis infection or vaccination.

Whether cytokines can influence the adaptive immune response by antigen-specific γδ T cells during infections or vaccinations remains unknown. We previously demonstrated that, during BCG/Mycobacterium tuberculosis (Mtb) infections, Th17-related cytokines markedly upregulated when phosphoantigen-specific Vγ2Vδ2 T cells expanded. In this study, we examined the involvement of Th17-related cytokines in the recall-like responses of Vγ2Vδ2 T cells following Mtb infection or vaccination against TB. Treatment with IL-17A/IL-17F or IL-22 expanded phosphoantigen 4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP)-stimulated Vγ2Vδ2 T cells from BCG-vaccinated macaques but not from naïve animals, and IL-23 induced greater expansion than the other Th17-related cytokines. Consistently, Mtb infection of macaques also enhanced the ability of IL-17/IL-22 or IL-23 to expand HMBPP-stimulated Vγ2Vδ2 T cells. When evaluating IL-23 signaling as a prototype, we found that HMBPP/IL-23-expanded Vγ2Vδ2 T cells from macaques infected with Mtb or vaccinated with BCG or Listeria ΔactA prfA*-ESAT6/Ag85B produced IL-17, IL-22, IL-2, and IFN-γ. Interestingly, HMBPP/IL-23-induced production of IFN-γ in turn facilitated IL-23-induced expansion of HMBPP-activated Vγ2Vδ2 T cells. Furthermore, HMBPP/IL-23-induced proliferation of Vγ2Vδ2 T cells appeared to require APC contact and involve the conventional and novel protein kinase C signaling pathways. These findings suggest that Th17-related cytokines can contribute to recall-like expansion and effector function of Ag-specific γδ T cells after infection or vaccination.

Preparation and characterization of diethoxy- and monoethoxy phosphylated ('aged') serine haptens and use in the production of monoclonal antibodies.

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the 'aged' form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form.

HMBPP-deficient Listeria mutant immunization alters pulmonary/systemic responses, effector functions, and memory polarization of Vγ2Vδ2 T cells.

Whereas infection or immunization of humans/primates with microbes coproducing HMBPP/IPP can remarkably activate Vγ2Vδ2 T cells, in vivo studies have not been done to dissect HMBPP- and IPP-driven expansion, pulmonary trafficking, effector functions, and memory polarization of Vγ2Vδ2 T cells. We define these phosphoantigen-host interplays by comparative immunizations of macaques with the HMBPP/IPP-coproducing Listeria ΔactA prfA* and HMBPP-deficient Listeria ΔactA ΔGCPE: prfA* mutant. The HMBPP-deficient ΔGCPE: mutant shows lower ability to expand Vγ2Vδ2 T cells in vitro than the parental HMBPP-producing strain but displays comparably attenuated infectivity or immunogenicity. Respiratory immunization of macaques with the HMBPP-deficient mutant elicits lower pulmonary and systemic responses of Vγ2Vδ2 T cells compared with the HMBPP-producing vaccine strain. Interestingly, HMBPP-deficient mutant reimmunization or boosting elicits enhanced responses of Vγ2Vδ2 T cells, but the magnitude is lower than that by HMBPP-producing listeria. HMBPP-deficient listeria differentiated fewer Vγ2Vδ2 T effector cells capable of coproducing IFN-γ and TNF-α and inhibiting intracellular listeria than HMBPP-producing listeria. Furthermore, HMBPP deficiency in listerial immunization influences memory polarization of Vγ2Vδ2 T cells. Thus, both HMBPP and IPP production in listerial immunization or infection elicit systemic/pulmonary responses and differentiation of Vγ2Vδ2 T cells, but a role for HMBPP is more dominant. Findings may help devise immune intervention.

Immunogenic and antioxidant effects of a pathogen-associated prenyl pyrophosphate in Anopheles gambiae.

Despite efficient vector transmission, Plasmodium parasites suffer great bottlenecks during their developmental stages within Anopheles mosquitoes. The outcome depends on a complex three-way interaction between host, parasite and gut bacteria. Although considerable progress has been made recently in deciphering Anopheles effector responses, little is currently known regarding the underlying microbial immune elicitors. An interesting candidate in this sense is the pathogen-derived prenyl pyrophosphate and designated phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), found in Plasmodium and most eubacteria but not in higher eukaryotes. HMBPP is the most potent stimulant known of human Vγ9Vδ2 T cells, a unique lymphocyte subset that expands during several infections including malaria. In this study, we show that Vγ9Vδ2 T cells proliferate when stimulated with supernatants from intraerythrocytic stages of Plasmodium falciparum cultures, suggesting that biologically relevant doses of phosphoantigens are excreted by the parasite. Next, we used Anopheles gambiae to investigate the immune- and redox- stimulating effects of HMBPP. We demonstrate a potent activation in vitro of all but one of the signaling pathways earlier implicated in the human Vγ9Vδ2 T cell response, as p38, JNK and PI3K/Akt but not ERK were activated in the A. gambiae 4a3B cell line. Additionally, both HMBPP and the downstream endogenous metabolite isopentenyl pyrophosphate displayed antioxidant effects by promoting cellular tolerance to hydrogen peroxide challenge. When provided in the mosquito blood meal, HMBPP induced temporal changes in the expression of several immune genes. In contrast to meso-diaminopimelic acid containing peptidoglycan, HMBPP induced expression of dual oxidase and nitric oxide synthase, two key determinants of Plasmodium infection. Furthermore, temporal fluctuations in midgut bacterial numbers were observed. The multifaceted effects observed in this study indicates that HMBPP is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.

In vivo gene delivery with L-tyrosine polyphosphate nanoparticles.

The concept of gene therapy is promising; however, the perceived risks and side effects associated with this technology have severely dampened the researchers' enthusiasm. Thus, the development of a nonviral gene vector without immunological effects and with high transfection efficiency is necessary. Currently, most nonviral vectors have failed to achieve the in vivo transfection efficiencies of viral vectors due to their toxicity, rapid clearance, and/or inappropriate release rates. Although our previous studies have successfully demonstrated the controlled-release of plasmid DNA (pDNA) polyplexes encapsulated into nanoparticles formulated with l-tyrosine polyphosphate (LTP-pDNA nanoparticles), the in vivo transfection capabilities and immunogenicity of this delivery system have yet to be examined. Thus, we evaluate LTP-pDNA nanoparticles in an in vivo setting via injection into rodent uterine tissue. Our results demonstrate through X-gal staining and immunohistochemistry of uterine tissue that transfection has successfully occurred after a nine-day incubation. In contrast, the results for the control nanoparticles show results similar to those of shams. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) from the injected tissues confirms the transfection in vivo. To examine the immunogenicity, the l-tyrosine polyphosphate (LTP) nanoparticles have been evaluated in a mouse model. No significant differences in the activation of the innate immune system are observed. These data provide the first report for the potential use of controlled-release nanoparticles formulated from an amino acid based polymer as an in vivo nonviral vector for gene therapy.

Multifunctional immune responses of HMBPP-specific Vγ2Vδ2 T cells in M. tuberculosis and other infections.

Vγ2Vδ2 T (also known as Vγ9Vδ2 T) cells exist only in primates, and in humans represent a major γδ T-cell sub-population in the total population of circulating γδ T cells. Results from recent studies suggest that while (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen from Mycobacterium tuberculosis (Mtb) and other microbes activates and expands primate Vγ2Vδ2 T cells, the Vγ2Vδ2 T-cell receptor (TCR) recognizes and binds to HMBPP on antigen-presenting cells (APC). In response to HMBPP stimulus, Vγ2Vδ2 TCRs array to form signaling-related nanoclusters or nanodomains during the activation of Vγ2Vδ2 T cells. Primary infections with HMBPP-producing pathogens drive the evolution of multieffector functional responses in Vγ2Vδ2 T cells, although Vγ2Vδ2 T cells display different patterns of responses during the acute and chronic phases of Mtb infection and in other infections. Expanded Vγ2Vδ2 T cells in primary Mtb infection can exhibit a broader TCR repertoire and a greater clonal response than previously assumed, with different distribution patterns of Vγ2Vδ2 T-cell clones in lymphoid and non-lymphoid compartments. Emerging in vivo data suggest that HMBPP activation of Vγ2Vδ2 T cells appears to impact other immune cells during infection.

Multieffector-functional immune responses of HMBPP-specific Vγ2Vδ2 T cells in nonhuman primates inoculated with Listeria monocytogenes ΔactA prfA*.

Although Listeria monocytogenes can induce systemic infection causing spontaneous abortion, septicemia, and meningitis, studies have not been performed to investigate human anti-L. monocytogenes immune responses, including those of Ag-specific Vγ2Vδ2 T cells, a dominant human γδ T cell subset. L. monocytogenes is the only pathogen known to possess both the mevalonate and non-mevalonate isoprenoid biosynthesis pathways that produce metabolic phosphates or phosphoantigens activating human Vγ2Vδ2 T cells, making it interesting to explore in vivo anti-L. monocytogenes immune responses of Vγ2Vδ2 T cells. In this study, we demonstrated that subclinical systemic L. monocytogenes infection of rhesus macaques via parenteral inoculation or vaccination with an attenuated Listeria strain induced multieffector-functional immune responses of phosphoantigen-specific Vγ2Vδ2 T cells. Subclinical systemic infection and reinfection with attenuated L. monocytogenes uncovered the ability of Vγ2Vδ2 T cells to mount expansion and adaptive or recall-like expansion. Expanded Vγ2Vδ2 T cells could traffic to and accumulate in the pulmonary compartment and intestinal mucosa. Expanded Vγ2Vδ2 T cells could evolve into effector cells producing IFN-γ, TNF-α, IL-4, IL-17, or perforin after L. monocytogenes infection, and some effector Vγ2Vδ2 T cells could coproduce IL-17 and IFN-γ, IL-4 and IFN-γ, or TNF-α and perforin. Surprisingly, in vivo-expanded Vγ2Vδ2 T effector cells in subclinical L. monocytogenes infection could directly lyse L. monocytogenes-infected target cells and inhibit intracellular L. monocytogenes bacteria. Thus, we present the first demonstration, to our knowledge, of multieffector-functional Vγ2Vδ2 T cell responses against L. monocytogenes.

Synthesis and immunological evaluation of the 4-ß-glucoside of HMBPP.

HMBPP ((E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate) is a highly potent innate immunogen that stimulates human γδ T cells expressing the Vγ2Vδ2 T cell antigen receptor. To determine if glycoside conjugates of HMBPP retain activity, the 4-ß-glucoside and its acetylated homolog were synthesized and tested for their ability to stimulate γδ T cells. The glycoside HMBPP conjugate stimulated human γδ T cells with an EC(50) of 78nM. The tetraacetyl glycoside HMBPP conjugate was also active (EC(50)=360nM). The two isomeric mono-ß-glucosides of the parent (E)-2-methylbut-2-ene-1,4-diol, however, were not active. Thus, HMBPP glycosylated at the 4-OH position stimulates γδ T cells as long as the pyrophosphate moiety is present.

An assessment of common marmoset (Callithrix jacchus) γ9(+) T cells and their response to phosphoantigen in vitro.

γ9δ2 T cells are a primate-specific γδ T cell subtype that expand and become activated during infection, responding directly to phosphoantigens which are by-products of essential metabolic pathways in both bacteria and mammals. Analogues of natural phosphoantigens have been developed as potential immunotherapeutics for treatment of tumours and infectious diseases. Several non-human primate models have been used in preclinical studies, however, little is known about marmoset γ9δ2 T cell responses. We identified γ9(+) T cells in various tissues in the marmoset and determined that these cells respond to phosphoantigen in a similar manner to human γ9δ2 T cells in vitro. Both human γ9δ2 T cells and marmoset γ9(+) T cells were able to reduce growth of the intracellular bacterium Burkholderia pseudomallei in vitro following expansion with phosphoantigen. This suggests that the marmoset is an appropriate model for examining the immunotherapeutic potential of compounds which target γ9δ2 T cells.

Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides.

Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation.

Differentiation, distribution and gammadelta T cell-driven regulation of IL-22-producing T cells in tuberculosis.

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vgamma2Vdelta2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNgamma-producing Vgamma2Vdelta2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNgamma neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vgamma2Vdelta2 T-cell-driven IFNgamma-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vgamma2Vdelta2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB.

Gammadelta T cell immune manipulation during chronic phase of simian-human immunodeficiency virus infection [corrected] confers immunological benefits.

Vgamma2Vdelta2 T cells, a major human gammadelta T cell subset, recognize the phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by mycobacteria and some opportunistic pathogens, and they contribute to innate/adaptive/homeostatic and anticancer immunity. As initial efforts to explore Vgamma2Vdelta2 T cell-based therapeutics against HIV/AIDS-associated bacterial/protozoal infections and neoplasms, we investigated whether a well-defined HMBPP/IL-2 therapeutic regimen could overcome HIV-mediated immune suppression to massively expand polyfunctional Vgamma2Vdelta2 T cells, and whether such activation/expansion could impact AIDS pathogenesis in simian HIV (SHIV)-infected Chinese rhesus macaques. While HMBPP/IL-2 coadministration during acute or chronic phase of SHIV infection induced massive activation/expansion of Vgamma2Vdelta2 T cells, the consequences of such activation/expansions were different between these two treatment settings. HMBPP/IL-2 cotreatment during acute SHIV infection did not prevent the increases in peak and set-point viral loads or the accelerated disease progression seen with IL-2 treatment alone. In contrast, HMBPP/IL-2 cotreatment during chronic infection did not exacerbate disease, and more importantly it could confer immunological benefits. Surprisingly, although viral antigenic loads were not increased upon HMBPP/IL-2 cotreatment during chronic SHIV infection, HMBPP activation of Vgamma2Vdelta2 T cells boosted HIV Env-specific Ab titers. Such increases in Abs were sustained for >170 days and were immediately preceded by increased production of IFN-gamma, TNF-alpha, IL-4, and IL-10 during peak expansion of Vgamma2Vdelta2 T cells displaying memory phenotypes, as well as the short-term increased effector function of Vgamma2Vdelta2 T cells and CD4(+) and CD8(+) alphabeta T cells producing antimicrobial cytokines. Thus, HMBPP/Vgamma2Vdelta2 T cell-based intervention may potentially be useful for combating neoplasms and HMBPP-producing opportunistic pathogens in chronically HIV-infected individuals.

Photoaffinity antigens for human gammadelta T cells.

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.

In vitro cytotoxicity of polyphosphoester as a novel injectable alveolar replacement material.

The aim of this study was to investigate the in vitro cytotoxicity of polyphosphoester polymer used as a novel injectable alveolar bone substitutes for controlled delivery of tetracycline. Cell culture medium was exposed to the polymer (0.01-10 mg/mL) for 24 h. The L-929 mouse fibroblasts were then exposed to the treated cell culture medium for 24 h. Finally, cell viability and growth were assessed by using MTT assay and Alamar Blue assay. No significant cytotoxicity of the polyphosphoester against L-929 mouse fibroblasts was observed at a concentration up to 10 mg/mL (P>0.05). The two evaluation methods showed no significant differences (P>0.05). This study suggests that polyphosphoester does not demonstrate any significant toxic effects to cells in vitro and has the potential to be used both as a medical device and as scaffolds in tissue engineering applications.