A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44.

Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5-7, graft-versus-host disease8 and inflammatory bowel disease9,10.

Diverse cytomegalovirus US11 antagonism and MHC-A evasion strategies reveal a tit-for-tat coevolutionary arms race in hominids.

Recurrent, ancient arms races between viruses and hosts have shaped both host immunological defense strategies as well as viral countermeasures. One such battle is waged by the glycoprotein US11 encoded by the persisting human cytomegalovirus. US11 mediates degradation of major histocompatibility class I (MHC-I) molecules to prevent CD8+ T-cell activation. Here, we studied the consequences of the arms race between US11 and primate MHC-A proteins, leading us to uncover a tit-for-tat coevolution and its impact on MHC-A diversification. We found that US11 spurred MHC-A adaptation to evade viral antagonism: In an ancestor of great apes, the MHC-A A2 lineage acquired a Pro184Ala mutation, which confers resistance against the ancestral US11 targeting strategy. In response, US11 deployed a unique low-complexity region (LCR), which exploits the MHC-I peptide loading complex to target the MHC-A2 peptide-binding groove. In addition, the global spread of the human HLA-A*02 allelic family prompted US11 to employ a superior LCR strategy with an optimally fitting peptide mimetic that specifically antagonizes HLA-A*02. Thus, despite cytomegaloviruses low pathogenic potential, the increasing commitment of US11 to MHC-A has significantly promoted diversification of MHC-A in hominids.

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

Targeting of a natural killer cell receptor family by a viral immunoevasin.

Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the 'immunoevasin' m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer. The helical stalks of Ly49H lay centrally across the m157 platform, whereas its lectin domain was not required for recognition. Instead, m157 targeted an 'aromatic peg motif' present in stalks of both activating and inhibitory receptors of the Ly49 family, and substitution of this motif abrogated binding. Furthermore, ligation of m157 to Ly49H or Ly49C resulted in intracellular signaling. Accordingly, m157 has evolved to 'tackle the legs' of a family of NK cell receptors.

Assembly and Dynamics of the Bacterial Flagellum.

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45-50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

Time for bacteria to slow down.

The speed of the bacterial flagellar motor is thought to be regulated by structural changes in the motor. Two new studies, Boehm et al. (2010) in this issue and Paul et al. (2010) in Molecular Cell, now show that cyclic di-GMP also regulates flagellar motor speed through interactions between the cyclic di-GMP binding protein YcgR and the motor proteins.

Recognition of the nonclassical MHC class I molecule H2-M3 by the receptor Ly49A regulates the licensing and activation of NK cells.

The development and function of natural killer (NK) cells is regulated by the interaction of inhibitory receptors of the Ly49 family with distinct peptide-laden major histocompatibility complex (MHC) class I molecules, although whether the Ly49 family is able bind to other MHC class I-like molecules is unclear. Here we found that the prototypic inhibitory receptor Ly49A bound the highly conserved nonclassical MHC class I molecule H2-M3 with an affinity similar to its affinity for H-2D(d). The specific recognition of H2-M3 by Ly49A regulated the 'licensing' of NK cells and mediated 'missing-self' recognition of H2-M3-deficient bone marrow. Host peptide-H2-M3 was required for optimal NK cell activity against experimental metastases and carcinogenesis. Thus, nonclassical MHC class I molecules can act as cognate ligands for Ly49 molecules. Our results provide insight into the various mechanisms that lead to NK cell tolerance.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor.

Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.

Motile ghosts of the halophilic archaeon, Haloferax volcanii.

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

The cryo-EM structure of the endocytic receptor DEC-205.

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine-guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

Impacts of Cognitive Behavioral Therapy for Insomnia and Pain on Sleep in Women with Gynecologic Malignancies: A Randomized Controlled Trial.

Insomnia is an adverse cancer outcome impacting mood, pain, quality of life, and mortality in cancer patients. Cognitive Behavioral Therapy (CBT) is an evidence-based treatment for diverse psychophysiological disorders, including pain and insomnia. Primarily studied in breast cancer, there is limited research on CBT within gynecology oncology. This study examined CBT effects on subjective and behavioral sleep outcomes: Sleep Efficiency (SE), Sleep Quality (SQ), Total Wake Time (TWT), Sleep Onset Latency (SOL), and Wake After Sleep Onset (WASO). Thirty-five women with insomnia status/post-surgery for gynecologic cancer were randomized to CBT for insomnia and pain (CBTi.p., N = 18) or Psychoeducation (N = 17). Sleep was assessed via sleep diaries and wrist-worn actigraphy at baseline (T1), post-intervention (T2), and two-month follow-up (T3). Intent-to-treat analyses utilizing mixed linear modeling examined longitudinal group differences on sleep controlling for age and advanced cancer. All participants demonstrated improved (1) subjective SE (0.5, p < .01), SOL (-1.2, p < .01), TWT (-1.2, p < .01), and (2) behavioral SE (0.1, p = .02), TWT (-1.2, p = .03), WASO (-0.8, p < .01) across time. Group-level time trends were indicative of higher subjective SE (6.8, p = .02), lower TWT (-40.3, p = .01), and lower SOL (-13.0, p = .05) in CBTi.p. compared to Psychoeducation. Supplemental analyses examining clinical significance and acute treatment effects demonstrated clinical improvements in SE (T1), TWT (T2, T3), and SOL (T3). Remaining effects were not significant. Despite lacking power to detect interaction effects, CBTi.p. clinically improved sleep in women with gynecologic cancers and insomnia during the active treatment phase. Future research will focus on developing larger trials within underserved populations.

Effect of Reaction Media on Grafting Hydrophobic Polymers from Cellulose Nanocrystals via Surface-Initiated Atom-Transfer Radical Polymerization.

Hydrophobic polymer-grafted cellulose nanocrystals (CNCs) were produced via surface-initiated atom-transfer radical polymerization (SI-ATRP) in two different solvents to examine the role of reaction media on the extent of surface modification. Poly(butyl acrylate)-grafted CNCs were synthesized in either dimethylformamide (DMF) (D-PBA-g-CNCs) or toluene (T-PBA-g-CNCs) alongside a free polymer from a sacrificial initiator. The colloidal stability of unmodified CNCs, initiator-modified CNCs, and PBA-g-CNCs in water, DMF, and toluene was evaluated by optical transmittance. The enhanced colloidal stability of initiator-modified CNCs in DMF led to improved accessibility to initiator groups during polymer grafting; D-PBA-g-CNCs had 30 times more grafted chains than T-PBA-g-CNCs, determined by thermogravimetric and elemental analysis. D-PBA-g-CNCs dispersed well in toluene and were hydrophobic with a water contact angle of 124° (for polymer grafts > 13 kDa) compared to 25° for T-PBA-g-CNCs. The cellulose crystal structure was preserved, and individual nanoparticles were retained when grafting was carried out in either solvent. This work highlights that optimizing CNC colloidal stability prior to grafting is more crucial than solvent-polymer compatibility to obtain high graft densities and highly hydrophobic CNCs via SI-ATRP.

Cellulose Nanocrystal (CNC)-Latex Nanocomposites: Effect of CNC Hydrophilicity and Charge on Rheological, Mechanical, and Adhesive Properties.

Cellulose nanocrystals (CNCs), a sustainable nanomaterial, are in situ incorporated into emulsion-based pressure-sensitive adhesives (PSAs). Commercially available CNCs with different surface hydrophilicity and surface charge (CNC101 and CNC103 from CelluForce) are used to explore their role in PSA property modification. Viscosity measurements and atomic force microscopy reveal differences in degree of association between the CNCs and the latex particles depending on the surface properties of the CNCs. The more hydrophilic and higher surface charge CNCs (CNC101) show less association with the latex particles. Dynamic strain sweep tests are used to analyze the strain-softening of the nanocomposites based on CNC type and loading. The CNC101 nanocomposites soften at lower strains than their CNC103 counterparts. This behavior is confirmed via dynamic frequency tests and modeling of the nanocomposites' storage moduli, which suggest the formation of CNC aggregates of, on average, 3.8 CNC101 and 1.3 CNC103 nanoparticles. Finally, PSA properties, i.e., tack, peel strength, and shear strength, simultaneously increase upon addition of both CNC types, although to different extents. The relationship between the PSA properties and CNC surface properties confirms that the less hydrophilic CNCs lead to improved CNC dispersion in the PSA films and therefore, enhance PSA properties.

How latex film formation and adhesion at the nanoscale correlate to performance of pressure sensitive adhesives with cellulose nanocrystals.

Emulsion polymerized latex-based pressure-sensitive adhesives (PSAs) are more environmentally benign because they are synthesized in water but often underperform compared to their solution polymerized counterparts. Studies have shown a simultaneous improvement in the tack, and peel and shear strength of various acrylic PSAs upon the addition of cellulose nanocrystals (CNCs). This work uses atomic force microscopy (AFM) to examine the role of CNCs in (i) the coalescence of hydrophobic 2-ethyl hexyl acrylate/n-butyl acrylate/methyl methacrylate (EHA/BA/MMA) latex films and (ii) as adhesion modifiers over multiple length scales. Thin films with varying solids content and CNC loading were prepared by spin coating. AFM revealed that CNCs lowered the solids content threshold for latex particle coalescence during film formation. This improved the cohesive strength of the films, which was directly reflected in the increased shear strength of the EHA/BA/MMA PSAs with increasing CNC loading. Colloidal probe AFM indicated that the nano-adhesion of thicker continuous latex films increased with CNC loading when measured over small contact areas where the effect of surface roughness was negligible. Conversely, the beneficial effects of the CNCs on macroscopic PSA tack and peel strength were outweighed by the effects of increased surface roughness with increasing CNC loading over larger surface areas. This highlights that CNCs can improve both cohesive and adhesive PSA properties; however, the effects are most pronounced when the CNCs interact favourably with the latex polymer and are uniformly dispersed throughout the adhesive film. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Structural basis for the recognition of nectin-like protein-5 by the human-activating immune receptor, DNAM-1.

Nectin and nectin-like (Necl) adhesion molecules are broadly overexpressed in a wide range of cancers. By binding to these adhesion molecules, the immunoreceptors DNAX accessory molecule-1 (DNAM-1), CD96 molecule (CD96), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) play a crucial role in regulating the anticancer activities of immune effector cells. However, within this axis, it remains unclear how DNAM-1 recognizes its cognate ligands. Here, we determined the structure of human DNAM-1 in complex with nectin-like protein-5 (Necl-5) at 2.8 Å resolution. Unexpectedly, we found that the two extracellular domains (D1-D2) of DNAM-1 adopt an unconventional "collapsed" arrangement that is markedly distinct from those in other immunoglobulin-based immunoreceptors. The DNAM-1/Necl-5 interaction was underpinned by conserved lock-and-key motifs located within their respective D1 domains, but also included a distinct interface derived from DNAM-1 D2. Mutation of the signature DNAM-1 "key" motif within the D1 domain attenuated Necl-5 binding and natural killer cell-mediated cytotoxicity. Altogether, our results have implications for understanding the binding mode of an immune receptor family that is emerging as a viable candidate for cancer immunotherapy.

Prevalence and correlates of sleep apnea among US Veterans with chronic kidney disease.

The prevalence and correlates of sleep apnea (SA) among Veterans with chronic kidney disease (CKD), a population at high risk of both SA and CKD, are unknown. We performed a cross-sectional analysis of 248 Veterans (18-89 years) selected only for presence of moderate to severe CKD. All participants underwent full, unattended polysomnography, measurement of renal function and a sleepiness questionnaire. Logistic regression with backward selection was used to identify predictors of prevalent SA (apnea-hypopnea index [AHI, ≥15 events/hr] and prevalent nocturnal hypoxia [NH, % of total sleep time spent at <90% oxygen saturation]). The mean age of our cohort was 73.2 ± 9.6 years, 95% were male, 78% were Caucasian and the mean body mass index (BMI) was 30.3 ± 4.8 kg/m2 . The prevalence of SA was 39%. There was no difference in daytime sleepiness among those with and without SA. In the final model, older age, higher BMI and diabetes mellitus (DM) were associated with higher odds of SA, after controlling for age, BMI, race and sex. Higher BMI, DM, unemployed/retired status, current smoking and higher serum bicarbonate level were associated with prevalent NH. To sum, SA was common among Veterans with moderate to severe CKD. Although some traditional risk factors for SA were associated with SA in this population, sleepiness did not correlate with SA. Further study is needed to validate our findings and understand how best to address the high burden of SA among Veterans with CKD.

Effect of cognitive behavioural therapy on sleep and opioid medication use in adults with fibromyalgia and insomnia.

Sleep and opioid medications used to treat insomnia and chronic pain are associated with adverse side effects (falls and cognitive disturbance). Although behavioural treatments such as cognitive behavioral therapy for insomnia (CBT-I) and pain (CBT-P) improve sleep and clinical pain, their effects on sleep and opioid medication use are unclear. In this secondary analysis of published trial data, we investigated whether CBT-I and CBT-P reduced reliance on sleep/opioid medication in patients with fibromyalgia and insomnia (FMI). Patients with FMI (n = 113, Mage  = 53.0, SD = 10.9) completed 8 weeks of CBT-I (n = 39), CBT-P (n = 37) or waitlist control (WLC; n = 37). Participants completed 14 daily diaries at baseline, post-treatment and 6-month follow-up, assessing sleep and opioid medication usage. Multilevel modelling examined group by time effects on days of medication use. A significant interaction revealed CBT-P reduced the number of days of sleep medication use at post-treatment, but usage returned to baseline levels at follow-up. There were no other significant within- or between-group effects. CBT-P led to immediate reductions in sleep medication usage, despite lack of explicit content regarding sleep medication. CBT-I and CBT-P may be ineffective as stand-alone treatments for altering opioid use in FMI. Future work should explore CBT as an adjunct to other behavioural techniques for opioid reduction.