Structural basis for mouse LAG3 interactions with the MHC class II molecule I-Ab.

The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and ß2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

Strong damped Lyman-α absorption in young star-forming galaxies at redshifts 9 to 11.

Primordial neutral atomic gas, mostly composed of hydrogen, is the raw material for star formation in galaxies. However, there are few direct constraints on the amount of neutral atomic hydrogen (H i) in galaxies at early cosmic times. We analyzed James Webb Space Telescope (JWST) near-infrared spectroscopy of distant galaxies, at redshifts ≳8. From a sample of 12 galaxies, we identified three that show strong damped Lyman-α absorption due to H i in their local surroundings. The galaxies are located at spectroscopic redshifts of 8.8, 10.2, and 11.4, corresponding to 400 to 600 million years after the Big Bang. They have H i column densities ≳1022 cm-2, which is an order of magnitude higher than expected for a fully neutral intergalactic medium, and constitute a gas-rich population of young star-forming galaxies.

Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

The nature of an ultra-faint galaxy in the cosmic dark ages seen with JWST.

In the first billion years after the Big Bang, sources of ultraviolet (UV) photons are believed to have ionized intergalactic hydrogen, rendering the Universe transparent to UV radiation. Galaxies brighter than the characteristic luminosity L* (refs. 1,2) do not provide enough ionizing photons to drive this cosmic reionization. Fainter galaxies are thought to dominate the photon budget; however, they are surrounded by neutral gas that prevents the escape of the Lyman-α photons, which has been the dominant way to identify them so far. JD1 was previously identified as a triply-imaged galaxy with a magnification factor of 13 provided by the foreground cluster Abell 2744 (ref. 3), and a photometric redshift of z ≈ 10. Here we report the spectroscopic confirmation of this very low luminosity (≈0.05 L*) galaxy at z = 9.79, observed 480 Myr after the Big Bang, by means of the identification of the Lyman break and redward continuum, as well as multiple ≳4σ emission lines, with the Near-InfraRed Spectrograph (NIRSpec) and Near-InfraRed Camera (NIRCam) instruments. The combination of the James Webb Space Telescope (JWST) and gravitational lensing shows that this ultra-faint galaxy (MUV = -17.35)-with a luminosity typical of the sources responsible for cosmic reionization-has a compact (≈150 pc) and complex morphology, low stellar mass (107.19 M⊙) and subsolar (≈0.6 Z⊙) gas-phase metallicity.

LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition.

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.

DksA plays an essential role in regulating the virulence of Borrelia burgdorferi.

The RNA polymerase-binding protein DksA, together with the alarmone nucleotides (p)ppGpp, mediates the stringent response to nutrient starvation in Borrelia burgdorferi. To date, the contribution of DksA to B. burgdorferi infection remains unknown. We report here that DksA is essential for B. burgdorferi to infect a mammalian host. dksA expression was highly induced during infection. Moreover, a dksA-deficient mutant was incapable of infecting mice. The mutant displayed growth defects when cultured in vitro and resistance to osmotic pressure was markedly reduced. These phenotypes were fully restored to those of the wild type when dksA mutation was complemented. We further showed that DksA controlled the expression of virulence-associated lipoprotein OspC, likely via the central alternative sigma factor RpoS. Synthesis of RpoS was abolished in the dksA mutant, but rpoS transcription remained unaffected. Additionally, we found that the expression of clpX, clpA, clpP, and clpP2 was significantly increased in the mutant, suggesting that DksA may post-transcriptionally regulate rpoS expression via its effect on ClpXP and/or ClpAP proteases. These combined data demonstrate that DksA regulates B. burgdorferi virulence at least partially through its influence on RpoS and OspC. This study thus elucidates that, in addition to function as a stringent response regulator, DksA promotes the transcription and/or translation of genes contributing to B. burgdorferi infectivity.

The Lon-1 Protease Is Required by Borrelia burgdorferi To Infect the Mammalian Host.

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. In addition, B. burgdorferi encodes a second Lon homolog called Lon-1. Recent studies suggest that Lon-1 may function differently from the prototypical Lon protease. However, the function of Lon-1 in B. burgdorferi biology remains virtually unknown. Particularly, the contribution of Lon-1 to B. burgdorferi fitness and infection remains hitherto unexplored. Herein, we show that Lon-1 plays a critical role for the infection of B. burgdorferi in a mammalian host. We found that lon-1 was highly expressed during animal infection, implying an important function of this protein in bacterial infection. We further generated a lon-1 deletion mutant and an isogenic complemented strain. Relative to that of the wild-type strain, the infectivity of the mutant was severely attenuated in a murine infection model. Our data also showed that the mutant displayed growth defects in regular BSK-II medium. Furthermore, bacterial resistance to osmotic stress was markedly reduced when lon-1 was inactivated. When exposed to tert-butyl hydroperoxide, survival of the lon-1 mutant was impaired. In addition, production of several virulence factors, such as BosR, RpoS, and OspC, was elevated in the mutant. These phenotypes were restored when the lon-1 mutation was complemented. Finally, we created a lon-1(S714A) mutant and found that this mutant failed to infect mice, suggesting that the proteolytic activity of Lon-1 is essential for bacterial infection. Taken together, these results demonstrate that Lon-1 is required by B. burgdorferi to infect animal hosts and to cope with environmental stresses.

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host.

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.

Retrospective evaluation of the association between admission blood glucose and l-lactate concentrations in ponies and horses with gastrointestinal disease (2008-2016): 545 cases.

OBJECTIVES: A recent study described increased l-lactate concentrations in ponies with gastrointestinal disease compared to horses, but blood glucose (BG) concentrations were not considered. The study tested the hypothesis that BG and l-lactate concentrations are correlated in horses and ponies with gastrointestinal disease and that BG concentrations, not equid type (pony vs horse), are an independent predictor of L-lactate concentrations. It was further hypothesized that equid type was an independent predictor of BG concentrations. DESIGN: Retrospective study 2008-2016. SETTING: University teaching hospital. ANIMALS: Admission data from 545 animals (384 horses and 161 ponies) with gastrointestinal disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Data collected included signalment, clinicopathological findings on admission, and nature and location of the gastrointestinal lesion (strangulating vs non-strangulating and large vs small intestinal lesion). The association between admission blood l-lactate concentrations, equid type (pony or horse) and BG concentrations was investigated in a multivariable model. Admission l-lactate and BG concentrations were strongly correlated (n = 522; r = 0.63; P < 0.001). Ponies had significantly higher l-lactate (2.7 mmol/L (0.5-18.0 mmol/L) vs 1.4 mmol/L (0.3-19 mmol/L); P < 0.001) and BG concentrations than horses (8.4 mmol/L (4.2-24.4 mmol/L); 151 mg/dL (76-439 mg/dL) vs 6.9 mmol/L (3.4-26.8 mmol/L); 124 mg/dL (61-482 mg/dL); P < 0.001). In the multivariable analysis, l-lactate concentrations were significantly and positively associated with admission BG concentrations in all animals and also with equid type. For each millimole per liter (18 mg/dL) increase in BG, l-lactate concentrations increased by 7.9% (5.9, 9.9); P < 0.001. In comparison to ponies, l-lactate concentrations were decreased by 27.7% (37.4, 16.5); P < 0.001 in horses. Admission BG concentrations were significantly and positively associated with l-lactate concentrations in all animals. For each millimole per liter increase in l-lactate concentration, BG concentration increased by 6.2% (4.7, 7.6; P < 0.001). Admission BG concentrations were not associated with equid type. CONCLUSION: Admission BG concentrations and equid type are independent predictors of blood l-lactate concentrations in equids with gastrointestinal disease, but their relationship requires further investigation.

The CXXC Motifs Are Essential for the Function of BosR in Borrelia burgdorferi.

BosR, a Fur family member, is essential for the pathogenesis of the Lyme disease pathogen, Borrelia burgdorferi. Unlike typical Fur proteins in which DNA binding represses gene expression, binding of BosR to the rpoS promoter directly activates rpoS transcription in B. burgdorferi. However, virtually nothing is known concerning potential structural features and amino acid residues of BosR that are important for protein function and virulence regulation in B. burgdorferi. Particularly, it remains unknown what structural motifs or residues of BosR coordinate Zn, although previous analyses have indicated that the function of BosR may depend on Zn. To address these information gaps, we herein introduced mutations into four conserved cysteine residues in two putative CXXC motifs of BosR. Our data showed that the ability of BosR to bind Zn was dramatically reduced when the CXXC motifs were mutated. Moreover, we found that the two CXXC motifs contributed to the ability of BosR to form dimers. By using a trans-complementation genetic approach, we additionally demonstrated that both CXXC motifs of BosR were essential for in vivo gene expression regulation. Mutation of any of the four cysteines abolished the transcriptional activation of rpoS. In contrast to wild type BosR, each mutant protein was incapable of binding the rpoS promoter in electrophoretic mobility shift assays. The combined data strongly support that the two CXXC motifs and four cysteines constitute the structural site essential for Zn-coordination, protein dimerization, and the unique regulatory activity of BosR.

Lithium Accumulates in Neurogenic Brain Regions as Revealed by High Resolution Ion Imaging.

Lithium (Li) is a potent mood stabilizer and displays neuroprotective and neurogenic properties. Despite extensive investigations, the mechanisms of action have not been fully elucidated, especially in the juvenile, developing brain. Here we characterized lithium distribution in the juvenile mouse brain during 28 days of continuous treatment that result in clinically relevant serum concentrations. By using Time-of-Flight Secondary Ion Mass Spectrometry- (ToF-SIMS) based imaging we were able to delineate temporospatial lithium profile throughout the brain and concurrent distribution of endogenous lipids with high chemical specificity and spatial resolution. We found that Li accumulated in neurogenic regions and investigated the effects on hippocampal neurogenesis. Lithium increased proliferation, as judged by Ki67-immunoreactivity, but did not alter the number of doublecortin-positive neuroblasts at the end of the treatment period. Moreover, ToF-SIMS revealed a steady depletion of sphingomyelin in white matter regions during 28d Li-treatment, particularly in the olfactory bulb. In contrast, cortical levels of cholesterol and choline increased over time in Li-treated mice. This is the first study describing ToF-SIMS imaging for probing the brain-wide accumulation of supplemented Li in situ. The findings demonstrate that this technique is a powerful approach for investigating the distribution and effects of neuroprotective agents in the brain.