Distinct spatial immune microlandscapes are independently associated with outcomes in triple-negative breast cancer.

The utility of spatial immunobiomarker quantitation in prognostication and therapeutic prediction is actively being investigated in triple-negative breast cancer (TNBC). Here, with high-plex quantitative digital spatial profiling, we map and quantitate intraepithelial and adjacent stromal tumor immune protein microenvironments in systemic treatment-naïve (female only) TNBC to assess the spatial context in immunobiomarker-based prediction of outcome. Immune protein profiles of CD45-rich and CD68-rich stromal microenvironments differ significantly. While they typically mirror adjacent, intraepithelial microenvironments, this is not uniformly true. In two TNBC cohorts, intraepithelial CD40 or HLA-DR enrichment associates with better outcomes, independently of stromal immune protein profiles or stromal TILs and other established prognostic variables. In contrast, intraepithelial or stromal microenvironment enrichment with IDO1 associates with improved survival irrespective of its spatial location. Antigen-presenting and T-cell activation states are inferred from eigenprotein scores. Such scores within the intraepithelial compartment interact with PD-L1 and IDO1 in ways that suggest prognostic and/or therapeutic potential. This characterization of the intrinsic spatial immunobiology of treatment-naïve TNBC highlights the importance of spatial microenvironments for biomarker quantitation to resolve intrinsic prognostic and predictive immune features and ultimately inform therapeutic strategies for clinically actionable immune biomarkers.

Non-neutral evolution and reciprocal monophyly of two expressed Mhc class II B genes in Leach's storm-petrel.

The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach's storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7%). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species.

RNA-Seq alignment to individualized genomes improves transcript abundance estimates in multiparent populations.

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations.

Oxidative stress and aging: beyond correlation.

The oxidative stress theory of aging has become increasingly accepted as playing a role in the aging process, based primarily on a substantial accumulation of circumstantial evidence. In recent years, the hypothesis that mitochondrially generated reactive oxygen species play a role in organismal aging has been directly tested in both invertebrate and mammalian model systems. Initial results imply that oxidative damage, specifically the level of superoxide, does play a role in limiting the lifespans of invertebrates such as Drosophila melanogaster and Caenorhabditis elegans. In mammalian model systems, the effect of oxidative stress on lifespan is less clear, but there is evidence that antioxidant treatment protects against age-related dysfunction, including cognitive decline.