MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data

Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST

In Vivo Survival and Homeostatic Proliferation of Natural Killer Cells

While the specificity and development of natural killer (NK) cells have been intensely studied, little is known about homeostasis of the mature NK population. Here we show that mouse NK cells undergo homeostatic proliferation when transferred into NK-deficient Rag−/− γC−/− hosts. Normal NK functional activity is maintained during this process, although there are some changes in NK phenotype. Using cell sorting, we demonstrate that mature (Mac-1hi) NK cells undergo homeostatic proliferation in an NK-deficient environment, yet immature (Mac-1lo) NK cells also proliferate in such hosts. We find that mature NK cells survive but do not proliferate in hosts which possess an endogenous NK pool. However, we go on to show that mature NK survival is critically dependent on interleukin (IL)-15. Surprisingly, NK survival is also compromised after transfer of cells into IL-15Rα−/− mice, implying that IL-15 responsiveness by bystander cells is critical for NK maintenance. These data imply that, similar to T cells, homeostasis of the NK pool is much more dynamic than previously appreciated and this may be relevant to manipulation of NK cells for therapeutic purposes

Dissociating Markers of Senescence and Protective Ability in Memory T Cells

No unique transcription factor or biomarker has been identified to reliably distinguish effector from memory T cells. Instead a set of surface markers including IL-7Rα and KLRG1 is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with the tumor necrosis factor family member CD27 are frequently used to predict a memory T cell's ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime–boost vaccine strategies which repeatedly stimulate T cells with the aim of increasing memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, we report here that these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions

Self–class I MHC molecules support survival of naive CD8 T cells, but depress their functional sensitivity through regulation of CD8 expression levels

Previous studies have suggested that naive CD8 T cells require self-peptide–major histocompatability complex (MHC) complexes for maintenance. However, interpretation of such studies is complicated because of the involvement of lymphopenic animals, as lymphopenia drastically alters naive T cell homeostasis and function. In this study, we explored naive CD8 T cell survival and function in nonlymphopenic conditions by using bone marrow chimeric donors and hosts in which class I MHC expression is absent or limited to radiosensitive versus radioresistant cells. We found that long-term survival of naive CD8 T cells (but not CD4 T cells) was impaired in the absence of class I MHC. However, distinct from this effect, class I MHC deprivation also enhanced naive CD8 T cell responsiveness to low-affinity (but not high-affinity) peptide–MHC ligands. We found that this improved sensitivity was a consequence of up-regulated CD8 levels, which was mediated through a transcriptional mechanism. Hence, our data suggest that, in a nonlymphopenic setting, self-class I MHC molecules support CD8 T cell survival, but that these interactions also attenuate naive T cell sensitivity by dynamic tuning of CD8 levels

GA4GH: International policies and standards for data sharing across genomic research and healthcare.

The Global Alliance for Genomics and Health (GA4GH) aims to accelerate biomedical advances by enabling the responsible sharing of clinical and genomic data through both harmonized data aggregation and federated approaches. The decreasing cost of genomic sequencing (along with other genome-wide molecular assays) and increasing evidence of its clinical utility will soon drive the generation of sequence data from tens of millions of humans, with increasing levels of diversity. In this perspective, we present the GA4GH strategies for addressing the major challenges of this data revolution. We describe the GA4GH organization, which is fueled by the development efforts of eight Work Streams and informed by the needs of 24 Driver Projects and other key stakeholders. We present the GA4GH suite of secure, interoperable technical standards and policy frameworks and review the current status of standards, their relevance to key domains of research and clinical care, and future plans of GA4GH. Broad international participation in building, adopting, and deploying GA4GH standards and frameworks will catalyze an unprecedented effort in data sharing that will be critical to advancing genomic medicine and ensuring that all populations can access its benefits

The priming stimulus dictates senescence of memory T cells (46.15)

Abstract Immunization drives the proliferation and differentiation of effector CD8 T cells followed by a contraction phase after the peak of the T cell response. The survivors of this contraction phase differentiate into long-lived memory cells, which can provide life-long protection. Generating long lived T cell memory through vaccination holds great promise to provide protection in situations where antibody-inducing vaccines fail. Prime-boost strategies have been used to repeatedly stimulate T cells with the aim of increasing memory T cell frequency. However, repeated stimulation of the memory T cell pool leads to T cell senescence and loss of responsiveness. We sought to determine whether a prime-boost vaccination strategy can be developed that does not suffer from this drawback. We report that the extent of senescence at the memory stage is determined during the primary response. We developed a system that allows priming of CD8 T cells without acquisition of maximal effector function and show that the ensuing memory population is fully functional upon rechallenge. Importantly, these memory cells derived from minimal effector T cells have full access to non-lymphoid tissue and are less senescent upon rechallenge than memory cells that did acquire maximal effector markers and function during priming. Understanding how the nature of the priming stimulus shapes memory T cell differentiation allows situation dependent tailoring of vaccines to achieve maximal protection efficacy.</jats:p

Memory CD8 T cells that are bystander-activated during an infection control early pathogen replication in an innate-like manner (P4371)

Abstract Recent studies illustrate that during an infection the antigen non-specific memory CD8 T cell compartment is not simply an inert pool of cells, but can participate in the immune response. This bystander activation of memory CD8 T cells occurs in the absence of cognate antigen recognition, but the overall role of T cell receptor (TCR) mediated signals has not been defined. Furthermore, it is unknown if and how these cells contribute to the clearance of an infection. We measured the strength of TCR signals that bystander activated CD8 T cells (BA-CTLs) receive in vivo and found evidence of TCR signaling, albeit limited. Given this marginal contribution of the TCR, we sought to determine how BA-CTLs efficiently kill target cells. We demonstrate that BA-CTLs recognize and directly eliminate target cells in an innate-like, granzyme-dependent manner. We next selectively inhibited BA-CTL mediated killing in vivo during the course of an infection, which led to a significant defect in pathogen clearance. Our data suggest that BA-CTLs play a previously unappreciated, essential role in the early immune response to pathogens before the onset of a de-novo generated, primary CD8 T cell response.</jats:p