Showing papers by "Lucy Bird published in 2008"

Antibiotics from alligators

Abstract: The late Steve Irwin (‘The Crocodile Hunter’) would no doubt have been delighted to learn about recent research, presented at the American Chemical Society meeting (6–10 April) in New Orleans, USA, showing that proteins from alligator blood could pave the way for new antibiotics. Lancia Darville, at Louisiana State University and co-author of the study, had noticed that “...alligators tend to get into tussles and fights. They have torn limbs and scratches that are exposed to all of this bacteria in the water, yet they are never infected.” (USA Today, 8 April 2008.) To understand what might constitute this strong resistance to infection, the team collected blood samples from alligators after injecting them with a substance to stimulate their immune systems (BBC News, 6 April 2008). When researchers exposed 23 species of bacteria to alligator serum, all of them were destroyed, including MRSA (methicillin-resistant Staphylococcus aureus). By contrast, human serum destroyed only 8 of the bacterial strains (National Geographic News, 7 April 2008). Alligator blood proteins also showed antibiotic activity against the fungus Candida albicans and the virus HIV. Darville says, “That was a good indication that alligators must have some other additional proteins or some proteins that are overly expressed in their system that are either not present in ours or not overexpressed in ours” (USA Today). The scientists are now working to identify the specific peptides responsible for this antibacterial and antifungal activity. They estimate that alligator serum may contain at least four promising substances (ScienceDaily, 7 April 2008). “Once we sequence these peptides, we can obtain their chemical structure to potentially [create new] drugs.” (National Geographic News.) Co-author Mark Merchant thinks that “There’s a real possibility that you could be treated with an alligator blood product one day” (BBC News). Lucy Bird R e s e a R c h h i g h l i g h t s

Tumour immunotherapy: Little pig, little pig, let me come in

Abstract: nursery rhyme wolf could not blow down the little pigs’ house made of bricks. The wall of endothelial cells surrounding tumour-associated blood vessels can be equally impenetrable, preventing T cells from exiting the blood and destroying the tumour. A recent study in Nature Medicine identifies a new mechanism by which the tumour endothelium blocks T-cell homing and describes how this can be overcome to enhance the efficacy of tumour immunotherapy. To understand how the tumour endothelium might regulate T-cell homing, the authors compared gene expression by tumour endothelial cells (TECs) from human ovarian cancers with and without tumour-infiltrating lymphocytes (TILs). Numerous genes were found to be differentially expressed; of particular interest to the authors was endothelin B receptor (ETBR), which was expressed at higher levels by TECs from tumours lacking TILs than those with TILs. The ligand for ETBR, endothelin-1, is known to be produced at high levels by ovarian cancer cells and has a crucial role in regulating vascular homeostasis and permeability. Indeed, in an in vitro assay, the presence of endothelin-1 inhibited T-cell adhesion to endothelial cells (a step required for lymphocytes to migrate out of the bloodstream). However, the effect of endothelin-1 on adhesion could be neutralized by addition of the specific ETBR inhibitor BQ-788. Further studies showed that the endothelin-1– ETBR interaction suppressed T-cell adhesion by inhibiting the upregulation of expression and clustering of intercellular adhesion molecule 1 (ICAM1) on the endothelium and that nitric-oxide release was required for this effect. Accordingly, the presence of BQ-788 restored ICAM1 expression and reduced nitric-oxide release, thereby facilitating T-cell adhesion to endothelial cells in vitro. Next, the authors tested whether ETBR blockade by BQ-788 might have a beneficial effect on T-cell homing in mouse models of cancer and improve the efficacy of otherwise ineffective tumour immunotherapy protocols. In the two mouse models studied, systemic antitumour T-cell responses were induced by tumour vaccines (a tumour-cell-based vaccine and a DNA vaccine), but this did not delay the growth of inoculated tumour cells or prolong survival of the mice. However, when the vaccinated mice were treated with BQ-788, tumour growth was delayed and mouse survival increased. This beneficial effect of ETBR blockade by BQ-788 was shown to be the result of increased homing of tumour-specific T cells to the tumour and not due to the induction of higher numbers or activation of these cells. So, overriding the tumour-induced barrier function of the endothelium with pharmacological inhibitors, such as BQ-788, could prove to be a useful strategy to ensure that tumourspecific T cells that are induced in immunotherapeutic protocols gain sufficient access to the tumour.

T cells: Sharing through a synapse

Abstract: activated T cells in lymph nodes reveals that they engage with each other in dynamic clusters to share interleukin-2 (IL-2). To become activated, T cells engage with antigen-presenting cells (APCs), an interaction that is characterized by the formation of an immuno logical synapse. Once activated, T cells tend to have reduced motility and have been shown to associate in large clusters, but what might be the purpose of these T-cell clusters? To investigate this in detail, Krummel and colleagues used twophoton laser scanning microscopy to visualize the dynamics of activated T cells in vivo. Deep within the lymph nodes of mice that had received labelled antigen-specific T cells activated by subsequent immunization, the authors observed clusters of T cells. These clusters, which persisted for >30 minutes or were more transient, were highly dynamic, with T cells observed joining and leaving them. The clusters could form even if activation stimuli were not provided by APCs, which suggests that the capacity to self-aggregate is gained after T-cell activation but is not dependent on APCs. Further in vitro analysis showed that the activation-induced T-cell clustering is mediated by the integrin LFA1 (lymphocyte function-associated antigen 1), as fewer clusters were found in LFA1-deficient mice, and LFA1-specific antibody disrupted aggregates of wild-type T cells in vitro. In addition, electron microscopy analysis revealed that, similar to APC–T-cell interactions, multifocal synapses formed between the activated T cells. These synapses were characterized by the polarization of the microtubule-organizing complex and IL-2-containing vesicles towards the point of contact. This suggested that T-cell clustering might be of benefit to the T cells by facilitating the capture of IL-2 that is directionally secreted across the synapse. Indeed, synapse-engaged T cells were found to accumulate more IL-2 than those that were not engaged in synaptic contact and this resulted in substantially higher levels of phosphorylated STAT5 (signal transducer and activator of transcription 5), which is a transcription factor downstream of IL-2-receptor signalling. So, this study shows that T-cell activation is facilitated by the formation of T-cell clusters that allow polarized secretion of cytokines across a synapse. Lucy Bird

T-cell development: Galectin-1: the decision maker

Abstract: thymocyte rests on the affinity of its T-cell receptor (TCR) for peptide –MHC ligands, such that a highaffinity interaction with an agonist ligand leads to death (negative selection) and an intermediateaffinity interaction with a partial agonist ligand promotes survival (positive selection). But exactly how small differences in TCR affinity are functionally translated into cell fate remains unresolved. Now, Carrie Miceli and colleagues show that galectin-1 can act as a modulator of TCR signalling in thymocytes, opposing positive selection and promoting negative selection of conventional CD8+ T cells. Prompted by their previous studies indicating that the galactose-binding protein galectin-1 tunes TCR signalling in peripheral T cells, Liu et al. examined the role of galectin-1 in T-cell selection using the H-Y and OT-I TCR-transgenic mouse models. In the H-Y model, H-Y-specific thymocytes are positively selected in female mice but negatively selected in male mice, which express the agonist ligand male antigen H-Y. Deletion of the gene encoding galectin-1 in female mice was shown to promote positive selection of H-Y TCR+CD8+ T cells, as more CD8+ thymocytes and mature T cells were found in these mice than in their galectin-1sufficient counterparts. Galectin-1deficient H-Y TCR-transgenic male mice also had an increased number of H-Y TCR+CD8+ T cells compared with control male mice, suggesting that galectin-1 is needed for efficient negative selection. By contrast, gut-resident CD8αα+ intraepithelial lymphocytes, which are known to be positively selected in the thymus by agonist ligands, were present in reduced numbers in galectin-1deficient H-Y TCR-transgenic male mice, thereby consistent with a role for galectin-1 in promoting agonist-driven selection. The role of galectin-1 in opposing positive selection was also supported by studies of OT-I TCR-transgenic mice, in which galectin-1 deficiency was shown to enhance thymocyte positive selection driven by partial agonists. So how might galectin-1 modulate thymocyte selection thresholds? Assessment of the decay of peptide–MHC tetramer binding to TCR-transgenic thymocytes in the presence or absence of recombinant galectin-1 indicated that galectin-1 may promote agonist-driven selection by increasing binding of the TCR to agonist complexes. This hypothesis was tested by measuring the dynamics and levels of ERK (extracellularsignal-regulated kinase) activation in thymocytes exposed to CD3-specific antibody or agonist tetramer in vitro; low level sustained ERK activation is thought to result in positive selection, whereas rapid, transient ERK activation is thought to support negative selection. Consistent with the in vivo observations, the presence of galectin1 during thymocyte activation in vitro increased the intensity of the rapid and transient ERK activation that occurs on agonist-driven negative selection. By contrast, galectin-1 antagonized ERK activity in OT-I TCR+ thymocytes undergoing positive selection. Based on these observations, the authors propose that galectin-1 — possibly through the formation of glycolattice-based membrane microdomains — aids in the discrimination of TCR-directed cell-fate decisions. Lucy Bird

Parasite Immunity: How T cells see Toxo

Abstract: for protection against the intra­ cellular parasite Toxoplasma gondii. But the parasite­derived peptides that are recognized by the protective T cells have remained elusive. Now, Shastri and colleagues show that a single parasite peptide presented by the mouse MHC class I allele H2­Ld is the target of an immunodominant CD8+ T­cell response, and mice that fail to process this peptide correctly are susceptible to toxoplasmosis. T. gondii establishes productive infection within a specialized intracellular vacuole, existing either as rapidly growing tachyzoites or as bradyzoites in semidormant cysts in the brain or muscles. To identify parasite proteins that might access the MHC class I processing pathway and stimulate T­cell responses, the authors generated T. gondii­specific T­cell hybridomas and assessed their reactivity against H2­Ld­expressing cells transfected with a cDNA library produced from tachyzoites. One T­cell hybridoma responded to several cDNAs, which were all found to encode (full length or a truncated version of) the parasite protein GRA6. Further studies confirmed that GRA6 contained the T­cell­ stimulating peptide that bound to H2­Ld, a decapeptide termed HF10. To establish whether the HF10 peptide was naturally processed from GRA6 in T. gondii­infected cells, the authors tested the antigenic activity of peptide fractions derived from infected cells. Indeed, the peptide fraction that stimulated the T­cell hybridomas eluted at the same time point as synthetic HF10. In addition, HF10­specific CD8+ T cells could be detected in the spleen and brain of mice that were infected with T. gondii. Surprisingly, given that the large T. gondii genome encodes thousands of potential antigens, the entire T­cell population that was responding to the parasite was found to be specific for the HF10 peptide, which is consistent with HF10 being an immunodominant epitope. Finally, the observation that immunization with bone­marrow­derived dendritic cells pulsed with HF10, but not with an irrelevant control peptide, pro­ tected mice from an otherwise lethal challenge with T. gondii confirmed that the CD8+ T­cell response to this epitope confers protection against T. gondii infection. A clue to the natural processing of the HF10 peptide was provided by the finding that mice lacking ERAAP (endoplasmic reticulum amino­ peptidase associated with antigen processing; which is known to be important for customizing peptides for presentation by MHC molecules) were more susceptible to T. gondii infection. Cells from these mice were considerably compromised in their ability to activate T. gondii­specific T­cell hybridomas following infection owing to a defect in the generation of HF10–H2­Ld complexes, which was shown to depend on the proteasome and TAP (transporter associated with antigen processing), as well as ERAAP. So, these data indicate that the ability of exogenous or vacuolar antigens to access the cytosolic MHC class I processing pathway determines whether they serve as sources for peptide–MHC ligands that elicit CD8+ T­cell responses. Lucy Bird