A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8⁺ T cell epitope, NY-ESO-1_88–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1_157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1_88–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1_157–165. On the other hand, NY-ESO-1_157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A_26–35; whereas NY-ESO-1_88–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1_88–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18⁺ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1_88–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8⁺ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.     

Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.     

SERPINA1 PiZ and PiS Heterozygotes and Lung Function Decline in the SAPALDIA Cohort

Background

Severe alpha1-antitrypsin (AAT) deficiency is a strong risk factor for COPD. But the impact of gene variants resulting in mild or intermediate AAT deficiency on the longitudinal course of respiratory health remains controversial. There is indication from experimental studies that pro-inflammatory agents like cigarette smoke can interact with these variants and thus increase the risk of adverse respiratory health effects. Therefore, we tested the effect of the presence of a protease inhibitor (Pi) S or Z allele (PiMS and PiMZ) on the change in lung function in different inflammation-exposed subgroups of a large, population-based cohort study.

Methodology and Principal Findings

The SAPALDIA population includes over 4600 subjects from whom SERPINA1 genotypes for S and Z alleles, spirometry and respiratory symptoms at baseline and after 11 years follow-up, as well as proxies for inflammatory conditions, such as detailed smoking history, obesity and high sensitivity C-reactive protein (hs-CRP), were available. All analyses were performed by applying multivariate regression models. There was no overall unfavourable effect of PiMS or PiMZ genotype on lung function change. We found indication that PiZ heterozygosity interacted with inflammatory stimuli leading to an accelerated decline in measures in use as indices for assessing mild airway obstruction. Obese individuals with genotype PiMM had an average annual decline in the forced mid expiratory flow (ΔFEF25-75%) of 58.4 ml whereas in obese individuals with PiMZ it amounted to 92.2 ml (p = 0.03). Corresponding numbers for persistent smokers differed even more strongly (66.8 ml (PiMM) vs. 108.2 ml (PiMZ), p = 0.005). Equivalent, but less strong associations were observed for the change in the FEV1/FVC ratio.

Conclusions

We suggest that, in addition to the well established impact of the rare PiZZ genotype, one Z allele may be sufficient to accelerate lung function decline in population subgroups characterized by elevated levels of low grade inflammation.     

Recognizing biological motion and emotions from point-light displays in autism spectrum disorders

One of the main characteristics of Autism Spectrum Disorder (ASD) are problems with social interaction and communication. Here, we explored ASD-related alterations in 'reading' body language of other humans. Accuracy and reaction times were assessed from two observational tasks involving the recognition of 'biological motion' and 'emotions' from point-light displays (PLDs). Eye movements were recorded during the completion of the tests. Results indicated that typically developed-participants were more accurate than ASD-subjects in recognizing biological motion or emotions from PLDs. No accuracy differences were revealed on two control-tasks (involving the indication of color-changes in the moving point-lights). Group differences in reaction times existed on all tasks, but effect sizes were higher for the biological and emotion recognition tasks. Biological motion recognition abilities were related to a person's ability to recognize emotions from PLDs. However, ASD-related atypicalities in emotion recognition could not entirely be attributed to more basic deficits in biological motion recognition, suggesting an additional ASD-specific deficit in recognizing the emotional dimension of the point light displays. Eye movements were assessed during the completion of tasks and results indicated that ASD-participants generally produced more saccades and shorter fixation-durations compared to the control-group. However, especially for emotion recognition, these altered eye movements were associated with reductions in task-performance.     

Pax6 Directly Down-Regulates Pcsk1n Expression Thereby Regulating PC1/3 Dependent Proinsulin Processing

Background

Heterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing.

Methodology/Principal Findings

Using quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity.

Conclusions/Significance

Collectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.     

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11b(hi) DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.     

Foraging behavior and success of a mesopelagic predator in the northeast Pacific Ocean: insights from a data-rich species, the northern elephant seal

The mesopelagic zone of the northeast Pacific Ocean is an important foraging habitat for many predators, yet few studies have addressed the factors driving basin-scale predator distributions or inter-annual variability in foraging and breeding success. Understanding these processes is critical to reveal how conditions at sea cascade to population-level effects. To begin addressing these challenging questions, we collected diving, tracking, foraging success, and natality data for 297 adult female northern elephant seal migrations from 2004 to 2010. During the longer post-molting migration, individual energy gain rates were significant predictors of pregnancy. At sea, seals focused their foraging effort along a narrow band corresponding to the boundary between the sub-arctic and sub-tropical gyres. In contrast to shallow-diving predators, elephant seals target the gyre-gyre boundary throughout the year rather than follow the southward winter migration of surface features, such as the Transition Zone Chlorophyll Front. We also assessed the impact of added transit costs by studying seals at a colony near the southern extent of the species' range, 1,150 km to the south. A much larger proportion of seals foraged locally, implying plasticity in foraging strategies and possibly prey type. While these findings are derived from a single species, the results may provide insight to the foraging patterns of many other meso-pelagic predators in the northeast Pacific Ocean.     

Humoral immune responses to a single allele PfAMA1 vaccine in healthy malaria-naïve adults

Plasmodium falciparum: apical membrane antigen 1 (AMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. The polymorphic nature of AMA1 may compromise vaccine induced protection. The humoral response induced by two dosages (10 and 50 μg) of a single allele AMA1 antigen (FVO) formulated with Alhydrogel, Montanide ISA 720 or AS02 was investigated in 47 malaria-na?ve adult volunteers. Volunteers were vaccinated 3 times at 4 weekly intervals and serum samples obtained four weeks after the third immunization were analysed for (i) Antibody responses to various allelic variants, (ii) Domain specificity, (iii) Avidity, (iv) IgG subclass levels, by ELISA and (v) functionality of antibody responses by Growth Inhibition Assay (GIA). About half of the antibodies induced by vaccination cross reacted with heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of the antibody response, but had only a marginal influence on specificity, avidity, domain recognition or subclass responses. The highest antibody responses were observed for AMA1 formulated with AS02. The Growth Inhibition Assay activity of the antibodies was proportional to the amount of antigen specific IgG and the functional capacity of the antibodies was similar for heterologous AMA1-expressing laboratory strains. Trial registration: ClinicalTrials.gov NCT00730782.