Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T cell receptor (TCR) contact residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both BCG vaccination and M. tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.

Control flow defenses against ROP either use strict, expensive, but strong protection against redirected RET instructions with shadow stacks, or much faster but weaker protections without. In this work we study the inherent overheads of shadow stack schemes. We find that the overhead is roughly 10% for a traditional shadow stack. We then design a new scheme, the parallel shadow stack, and show that its performance cost is significantly less: 3.5%. Our measurements suggest it will not be easy to improve performance on current x86 processors further, due to inherent costs associated with RET and memory load/store instructions. We conclude with a discussion of the design decisions in our shadow stack instrumentation, and possible lighter-weight alternatives.

The pathogenic agent responsible for the expanded repeat diseases, a group of neurodegenerative diseases that includes Huntington's disease is not yet fully understood. Expanded polyglutamine (polyQ) is thought to be the toxic agent in certain cases, however, not all expanded repeat disease genes can encode a polyQ sequence. Since a repeat-containing RNA intermediary is common to all of these diseases, hairpin-forming single-stranded RNA has been investigated as a potential common pathogenic agent. More recently, it has become apparent that most of the expanded repeat disease loci have transcription occurring from both strands, raising the possibility that the complementary repeat RNAs could form a double-stranded structure. In our investigation using Drosophila models of these diseases, we identified a fortuitous integration event that models bidirectional repeat RNA transcription with the resultant flies exhibiting inducible pathology. We therefore established further lines of Drosophila expressing independent complementary repeat RNAs and found that these are toxic. The Dicer pathway is essential for this toxicity and in neuronal cells accounts for metabolism of the high copy number (CAG.CUG)(100) double-stranded RNAs down to (CAG)(7) single-stranded small RNAs. We also observe significant changes to the microRNA profile in neurons. These data identify a novel pathway through which double-stranded repeat RNA is toxic and capable of eliciting symptoms common to neurodegenerative human diseases resulting from dominantly inherited expanded repeats.

The elderly are particularly susceptible to influenza A virus infections, with increased occurrence, disease severity and reduced vaccine efficacy attributed to declining immunity. Experimentally, the age-dependent decline in influenza-specific CD8+ T cell responsiveness reflects both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. In this study, we asked whether early priming limits the time-related attrition of immune competence. Though primary responses in aged mice were compromised, animals vaccinated at 6 weeks then challenged >20 months later had T-cell responses that were normal in magnitude. Both functional quality and the persistence of ‘preferred’ TCR clonotypes that expand in a characteristic immunodominance hierarchy were maintained following early priming. Similar to the early priming, vaccination at 22 months followed by challenge retained a response magnitude equivalent to young mice. However, late priming resulted in reduced TCRβ diversity in comparison with vaccination earlier in life. Thus, early priming was critical to maintaining individual and population-wide TCRβ diversity. In summary, early exposure leads to the long-term maintenance of memory T cells and thus preserves optimal, influenza-specific CD8+ T-cell responsiveness and protects against the age-related attrition of naïve T-cell precursors. Our study supports development of vaccines that prime CD8+ T-cells early in life to elicit the broadest possible spectrum of CD8+ T-cell memory and preserve the magnitude, functionality and TCR usage of responding populations. In addition, our study provides the most comprehensive analysis of the aged (primary, secondary primed-early and secondary primed-late) TCR repertoires published to date.

Natural selection acts on variation that is typically assumed to be genetic in origin. But epigenetic mechanisms, which are interposed between the genome and its environment, can create diversity independently of genetic variation. Epigenetic states can respond to environmental cues, and can be heritable, thus providing a means by which environmentally responsive phenotypes might be selectable independent of genotype. Here, we have tested the possibility that environment and selection can act together to increase the penetrance of an epigenetically determined phenotype. We used isogenic A vy mice, in which the epigenetic state of the A vy allele is sensitive to dietary methyl donors. By combining methyl donor supplementation with selection for a silent A vy allele, we progressively increased the prevalence of the associated phenotype in the population over five generations. After withdrawal of the dietary supplement, the shift persisted for one generation but was lost in subsequent generations. Our data provide the first demonstration that selection for a purely epigenetic trait can result in cumulative germline effects in mammals. These results present an alternative to the paradigm that natural selection acts only on genetic variation, and suggest that epigenetic changes could underlie rapid adaptation of species in response to natural environmental fluctuations.

No abstract

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an antigen-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of antigen during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate antigen OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent antigen-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with antigen occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.