Characterization of T-bet and eomes in peripheral human immune cells.

The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4(+) and CD8(+) T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells. The corrigendum regards data and text for the final figure of the manuscript, Figure 7: Subsequent analysis of T-bet levels in human lymphocytes comparing different permeabilization procedures (eBioscience FoxP3 transcription factor kit, BD Pharmingen Cytofix/Cytoperm) has revealed variable findings in the level of T-bet expression detected within certain lymphocyte populations. While this does not change our conclusions for the majority of the populations assessed in this study, B cells in particular show differences under these conditions. Specifically, permeabilization via the eBioscience FoxP3 transcription factor staining buffer set indicates that subpopulations of memory B cells express significantly higher levels of T-bet (MFI) compared to plasmablasts, and that plasmablasts express T-bet only at low levels. Subsequent RNA transcript analysis confirms that plasmablasts express T-bet RNA at a level comparable to naïve B cells. Together, in combination with fluorescence-minus-one and isotype control studies, these new findings suggest that subsets memory B cells, not plasmablasts, express the highest levels of T-bet in the B cell compartment and plasmablasts express T-bet at a lower frequency than is reported in Figure 7. Figure 7 Legend should read: (C) Histograms depicting T-bet expression levels in B-cells and NK cells from a representative donor. Histograms represent the following subsets: naïve B-cells (thick black line), memory B-cells (shaded gray), plasmablasts (thin black line), CD56bright NK cells (gray line), and CD56dim NK cells (shaded black). B-cell results section should be titled T-bet is predominantly expressed in mature memory B-cells and should read: While Eomes was undetectable in B-cells (data not shown), we found T-bet in ~10% of B-cells (Figure 7B). This T-bet expression was largely relegated to memory B-cells, with significantly lower amounts observed in transitional/immature B-cells, naïve B-cells, and plasmablasts (Figure 7B). Greater than 15% of memory B-cells expressed T-bet, a significantly higher frequency than that of all other B-cell populations, suggesting that T-bet may play a particularly important role in memory B-cell function. The discussion related to T-bet expression in plasmablasts should be reconsidered as follows: We found that T-bet is not significantly expressed in transitional/immature B-cells, naïve B-cells, and plasmablasts, but is highly expressed in subsets of memory-B cells. Reduced frequencies of T-bet expression in plasmablasts indicate a specific role for T-bet at the memory B-cell stage of development, which may no longer be necessary after further differentiation to the plasmablast stage. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

Long-Term Persistence of Exhausted CD8 T Cells in Chronic Infection Is Regulated by MicroRNA-155

Persistent viral infections and tumors drive development of exhausted T (TEX) cells. In these settings, TEX cells establish an important host-pathogen or host-tumor stalemate. However, TEX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained TEX cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection. Genetic deficiency of miR-155 ablated CD8 T cell responses during chronic infection. Conversely, enhanced miR-155 expression promoted expansion and long-term persistence of TEX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal TEX cell subset. Transcriptional profiling identified coordinated control of cell signaling and transcription factor pathways, including the key AP-1 family member Fosl2. Overexpression of Fosl2 reversed the miR-155 effects, identifying a link between miR-155 and the AP-1 transcriptional program in regulating TEX cells. Thus, we identify a mechanism of miR-155 regulation of TEX cells and a key role for Fosl2 in T cell exhaustion. During persistent viral infections, exhausted T cells (TEX) erode quantitatively and qualitatively and therefore fail to provide protection. Stelekati et al. identified microRNA (miR)-155 as a key molecule that can enhance and sustain TEX responses long-term during chronic viral infection

AGN STORM 2. I. First results: A Change in the Weather of Mrk 817

We present the first results from the ongoing, intensive, multiwavelength monitoring program of the luminous Seyfert 1 galaxy Mrk 817. While this active galactic nucleus was, in part, selected for its historically unobscured nature, we discovered that the X-ray spectrum is highly absorbed, and there are new blueshifted, broad, and narrow UV absorption lines, which suggest that a dust-free, ionized obscurer located at the inner broad-line region partially covers the central source. Despite the obscuration, we measure UV and optical continuum reverberation lags consistent with a centrally illuminated Shakura–Sunyaev thin accretion disk, and measure reverberation lags associated with the optical broad-line region, as expected. However, in the first 55 days of the campaign, when the obscuration was becoming most extreme, we observe a de-coupling of the UV continuum and the UV broad emission-line variability. The correlation recovered in the next 42 days of the campaign, as Mrk 817 entered a less obscured state. The short C IV and Lyα lags suggest that the accretion disk extends beyond the UV broad-line region. Unified

The Classical Nuclear Localization Signal Receptor, Importin-α, Is Required for Efficient Transition Through the G1/S Stage of the Cell Cycle in Saccharomyces cerevisiae

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-α/Srp1, to the G2/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-α/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G1 to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G1/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G2/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-α mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G1/S cell cycle transition

Bioenergetic Insufficiencies Due to Metabolic Alterations Regulated by the Inhibitory Receptor PD-1 Are an Early Driver of CD8(+) T Cell Exhaustion

Dynamic reprogramming of metabolism is essential for T cell effector function and memory formation. However, the regulation of metabolism in exhausted CD8(+) T (Tex) cells is poorly understood. We found that during the first week of chronic lymphocytic choriomeningitis virus (LCMV) infection, before severe dysfunction develops, virus-specific CD8(+) T cells were already unable to match the bioenergetics of effector T cells generated during acute infection. Suppression of T cell bioenergetics involved restricted glucose uptake and use, despite persisting mechanistic target of rapamycin (mTOR) signaling and upregulation of many anabolic pathways. PD-1 regulated early glycolytic and mitochondrial alterations and repressed transcriptional coactivator PGC-1α. Improving bioenergetics by overexpression of PGC-1α enhanced function in developing Tex cells. Therapeutic reinvigoration by anti-PD-L1 reprogrammed metabolism in a subset of Tex cells. These data highlight a key metabolic control event early in exhaustion and suggest that manipulating glycolytic and mitochondrial metabolism might enhance checkpoint blockade outcomes