Programmed Death (PD)-1-Deficient Mice Are Extremely Sensitive to Murine Hepatitis Virus Strain-3 (MHV-3) Infection

The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4+, CD8+ T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy

Bacteria Modulate the CD8+ T Cell Epitope Repertoire of Host Cytosol-Exposed Proteins to Manipulate the Host Immune Response

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested

Estimating the Fitness Cost of Escape from HLA Presentation in HIV-1 Protease and Reverse Transcriptase

Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified. In this study, we assess the replicative cost of mutations which are likely to escape presentation by HLA molecules in the region of HIV-1 protease and reverse transcriptase. Specifically, we combine computational approaches for prediction of in vitro replicative fitness and peptide binding affinity to HLA molecules. We find that mutations which impair binding to HLA-A molecules tend to have lower in vitro replicative fitness than mutations which do not impair binding to HLA-A molecules, suggesting that HLA-A escape mutations carry higher fitness costs than non-escape mutations. We argue that the association between fitness and HLA-A binding impairment is probably due to an intrinsic cost of escape from HLA-A molecules, and these costs are particularly strong for HLA-A alleles associated with efficient virus control. Counter-intuitively, we do not observe a significant effect in the case of HLA-B, but, as discussed, this does not argue against the relevance of HLA-B in virus control. Overall, this article points to the intriguing possibility that HLA-A molecules preferentially target more conserved regions of HIV-1, emphasizing the importance of HLA-A genes in the evolution of HIV-1 and RNA viruses in general

Whole-Genome Immunoinformatic Analysis of F. tularensis: Predicted CTL Epitopes Clustered in Hotspots Are Prone to Elicit a T-Cell Response

The cellular arm of the immune response plays a central role in the defense against intracellular pathogens, such as F. tularensis. To date, whole genome immunoinformatic analyses were limited either to relatively small genomes (e.g. viral) or to preselected subsets of proteins in complex pathogens. Here we present, for the first time, an unbiased bacterial global immunoinformatic screen of the 1740 proteins of F. tularensis subs. holarctica (LVS), aiming at identification of immunogenic peptides eliciting a CTL response. The very large number of predicted MHC class I binders (about 100,000, IC50 of 1000 nM or less) required the design of a strategy for further down selection of CTL candidates. The approach developed focused on mapping clusters rich in overlapping predicted epitopes, and ranking these “hotspot” regions according to the density of putative binding epitopes. Limited by the experimental load, we selected to screen a library of 1240 putative MHC binders derived from 104 top-ranking highly dense clusters. Peptides were tested for their ability to stimulate IFNγ secretion from splenocytes isolated from LVS vaccinated C57BL/6 mice. The majority of the clusters contained one or more CTL responder peptides and altogether 127 novel epitopes were identified, of which 82 are non-redundant. Accordingly, the level of success in identification of positive CTL responders was 17–25 fold higher than that found for a randomly selected library of 500 predicted MHC binders (IC50 of 500 nM or less). Most proteins (ca. 2/3) harboring the highly dense hotspots are membrane-associated. The approach for enrichment of true positive CTL epitopes described in this study, which allowed for over 50% increase in the dataset of known T-cell epitopes of F. tularensis, could be applied in immunoinformatic analyses of many other complex pathogen genomes

10 years of mindlines: a systematic review and commentary

Background: In 2004, Gabbay and le May showed that clinicians generally base their decisions on mindlines—internalised and collectively reinforced tacit guidelines—rather than consulting written clinical guidelines. We considered how the concept of mindlines has been taken forward since. Methods: We searched databases from 2004 to 2014 for the term ‘mindline(s)’ and tracked all sources citing Gabbay and le May’s 2004 article. We read and re-read papers to gain familiarity and developed an interpretive analysis and taxonomy by drawing on the principles of meta-narrative systematic review. Results: In our synthesis of 340 papers, distinguished between authors who used mindlines purely in name (‘nominal’ view) sometimes dismissing them as a harmful phenomenon, and authors who appeared to have understood the term’s philosophical foundations. The latter took an ‘in-practice’ view (studying how mindlines emerge and spread in real-world settings), a ‘theoretical and philosophical’ view (extending theory) or a ‘solution focused’ view (exploring how to promote and support mindline development). We found that it is not just clinicians who develop mindlines: so do patients, in face-to-face and (potentially) online communities. Theoretical publications on mindlines have continued to challenge the rationalist assumptions of evidence-based medicine (EBM). Conventional EBM assumes a single, knowable reality and seeks to strip away context to generate universal predictive rules. In contrast, mindlines are predicated on a more fluid, embodied and intersubjective view of knowledge; they accommodate context and acknowledge multiple realities. When considering how knowledge spreads, the concept of mindlines requires us to go beyond the constraining notions of ‘dissemination’ and ‘translation’ to study tacit knowledge and the interactive human processes by which such knowledge is created, enacted and shared. Solution-focused publications described mindline-promoting initiatives such as relationship-building, collaborative learning and thought leadership. Conclusions: The concept of mindlines challenges the naïve rationalist view of knowledge implicit in some EBM publications, but the term appears to have been misunderstood (and prematurely dismissed) by some authors. By further studying mindlines empirically and theoretically, there is potential to expand EBM’s conceptual toolkit to produce richer forms of ‘evidence-based’ knowledge. We outline a suggested research agenda for achieving this goal