SignificanceExceptionally long-lived people such as supercentenarians tend to spend their entire lives in good health, implying that their immune system remains active to protect against infections and tumors. However, their immunological condition has been largely unexplored. We profiled thousands of circulating immune cells from supercentenarians at single-cell resolution and identified CD4 T cells that have cytotoxic features. This characteristic is very unique to supercentenarians, because generally CD4 T cells have helper, but not cytotoxic, functions under physiological conditions. We further profiled their T cell receptors and revealed that the cytotoxic CD4 T cells were accumulated through clonal expansion. The conversion of helper CD4 T cells to a cytotoxic variety might be an adaptation to the late stage of aging.

Finding and characterizing mRNAs, their transcription start sites (TSS), and their associated promoters is a major focus in post-genome biology. Mammalian cells have at least 5–10 magnitudes more TSS than previously believed, and deeper sequencing is necessary to detect all active promoters in a given tissue. Here, we present a new method for high-throughput sequencing of 5′ cDNA tags—DeepCAGE: merging the Cap Analysis of Gene Expression method with ultra-high-throughput sequence technology. We apply DeepCAGE to characterize 1.4 million sequenced TSS from mouse hippocampus and reveal a wealth of novel core promoters that are preferentially used in hippocampus: This is the most comprehensive promoter data set for any tissue to date. Using these data, we present evidence indicating a key role for the Arnt2 transcription factor in hippocampus gene regulation. DeepCAGE can also detect promoters used only in a small subset of cells within the complex tissue.

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob͞cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4 -isothiocyanato-stilben-2,2 -disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.ion channel ͉ olfaction ͉ olfactory sensory neurons ͉ patch-clamp ͉ sensory coding

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

Supercentenarians, people who have reached 110 years of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from seven supercentenarians and five younger controls. We identified a marked increase of cytotoxic CD4 T-cells (CD4 CTLs) coupled with a substantial reduction of B-cells as a novel signature of supercentenarians. Furthermore, single-cell T-cell receptor sequencing of two supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15% to 35% of the entire CD4 T-cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.SignificanceExceptionally long-lived people such as supercentenarians tend to spend their entire lives in good health, implying that their immune system remains active to protect against infections and tumors. However, their immunological condition has been largely unexplored. We profiled thousands of circulating immune cells from supercentenarians at single-cell resolution, and identified a large number of CD4 T-cells that have cytotoxic features. This characteristic is very unique to supercentenarians, because generally CD4 T-cells have helper, but not cytotoxic, functions under physiological conditions. We further profiled their T-cell receptors, and revealed that the cytotoxic CD4 T-cells were accumulated through clonal expansion. The conversion of helper CD4 T-cells to a cytotoxic variety might be an adaptation to the late stage of aging.