Resident memory T cells (TRM cells) are an important first-line defense against respiratory pathogens, but the unique contributions of lung TRM cell populations to protective immunity and the factors that govern their localization to different compartments of the lung are not well understood. Here, we show that airway and interstitial TRM cells have distinct effector functions and that CXCR6 controls the partitioning of TRM cells within the lung by recruiting CD8 TRM cells to the airways. The absence of CXCR6 significantly decreases airway CD8 TRM cells due to altered trafficking of CXCR6−/− cells within the lung, and not decreased survival in the airways. CXCL16, the ligand for CXCR6, is localized primarily at the respiratory epithelium, and mice lacking CXCL16 also had decreased CD8 TRM cells in the airways. Finally, blocking CXCL16 inhibited the steady-state maintenance of airway TRM cells. Thus, the CXCR6/CXCL16 signaling axis controls the localization of TRM cells to different compartments of the lung and maintains airway TRM cells.

Summary Bacillus anthracis, the causative agent of anthrax disease, is lethal due to the actions of two exotoxins, anthrax lethal toxin (LT) and edema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occur through damage to distinct cell-types; while targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Surprisingly, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not appear to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.

Resident memory CD8 T (TRM) cells in the lung parenchyma (LP) and airways provide heterologous protection against influenza virus challenge. However, scant knowledge exists regarding factors necessary to establish and maintain lung CD8 TRM. Here we demonstrate that, in contrast to mechanisms described for other tissues, airway and LP CD8 TRM establishment requires cognate antigen recognition in the lung. Systemic effector CD8 T cells could be transiently pulled into the lung in response to localized inflammation, however these effector cells failed to establish tissue residency unless antigen was present in the pulmonary environment. The interaction of effector CD8 T cells with cognate antigen in the lung resulted in increased and prolonged expression of the tissue retention markers CD69 and CD103, and increased expression of the adhesion molecule VLA-1. The inability of localized inflammation alone to establish lung TRM resulted in decreased viral clearance and increased mortality following heterosubtypic influenza challenge, despite equal numbers of circulating memory CD8 T cells. These findings demonstrate that pulmonary antigen encounter is required for the establishment of lung CD8 TRM and may inform future vaccine strategies to generate robust cellular immunity against respiratory pathogens.

To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly 717 Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly 717 -to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1 −/− mice, eEF2 G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2 G717R/G717R mice showed milder phenotypes than OVCA1 −/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2 G717R/G717R /OVCA1 −/− double mutant mice displayed the milder phenotypes of the eEF2 G717R/G717R mice, suggesting that the embryonic lethality of OVCA1 −/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly 717 -to-Arg mutation cannot rescue this defect. E ukaryotic elongation factor 2 (eEF2) is a member of the GTPbinding translation elongation factor family, and an essential factor for protein synthesis and cell survival. eEF2 drives the GTP-dependent translocation of the nascent polypeptide chain from the A site to the P site of the ribosome and advances mRNA by three bases during the elongation cycle of protein synthesis (1). eEF2 is highly homologous in all eukaryotes. In fact, eEF2 of humans, rats, mice, hamsters, and other mammals have exactly the same amino acid sequence. Intriguingly, all eukaryotic eEF2 proteins contain a unique posttranslationally modified histidine residue termed diphthamide (2, 3). Diphthamide modification occurs after eEF2 is translated and is irreversible, marking the completion of the biosynthesis of eEF2.Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig. S1), thus inactivating eEF2, halting cellular protein synthesis, and causing cell death.Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective a...

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.

Cationic β-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of three binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component, which acts on certain intracellular targets, and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. Here we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different β-cyclodextrins (AmPrβCD and AMBnTβCD) in the PA(63), C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTβCD, demonstrated significantly stronger binding compared with AmPrβCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnTβCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrβCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration-independent, short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA(63), which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers.

Glycans within human lungs are recognized by many pathogens such as influenza A virus (IAV), yet little is known about their structures. Here we present the first analysis of the N- and O- and glycosphingolipid-glycans from total human lungs, along with histological analyses of IAV binding. The N-glycome of human lung contains extremely large complex-type N-glycans with linear poly-N-acetyllactosamine (PL) [-3Galβ1–4GlcNAcβ1-]n extensions, which are predominantly terminated in α2,3-linked sialic acid. By contrast, smaller N-glycans lack PL and are enriched in α2,6-linked sialic acids. In addition, we observed large glycosphingolipid (GSL)-glycans, which also consists of linear PL, terminating in mainly α2,3-linked sialic acid. Histological staining revealed that IAV binds to sialylated and non-sialylated glycans and binding is not concordant with respect to binding by sialic acid-specific lectins. These results extend our understanding of the types of glycans that may serve as binding sites for human lung pathogens.

Background: Anthrax toxin protective antigen (PA) forms heptameric or octameric oligomers after proteolytic activation. Results: We engineered two PA variants that form active octamers only when both versions are present. Conclusion: These PA variants enlarged the therapeutic window when used to target tumors compared with previous systems. Significance: This is the first method to generate a pure pool of octameric PA oligomer.