Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.     

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.     

Ecological and evolutionary consequences of spatial and temporal variation in pre-dispersal seed predation

Pre-dispersal seed predation may have important effects on population dynamics and trait evolution in plants. In this review, we first present a conceptual framework of the strength of pre-dispersal seed predation and its variation in space and time. We consider the interaction between plants and their seed predators to be “strong” when it affects plant population dynamics or causes changes in plant trait–fitness relationships, and “weak” when it has no such effects, and propose ways of how to adequately assess these effects. Second, we review the ecological literature between 1991 and 2005 to evaluate documented effects of pre-dispersal seed predation on plants and draw five major conclusions. (1) Pre-dispersal seed predation rates are usually low but sometimes high, and show a considerable variation in space and time. (2) Direct evidence suggests that pre-dispersal seed predation can have a significant effect on recruitment and plant population growth rate. Accumulating evidence of seed-limited recruitment suggests that such effects are common. (3) Pre-dispersal seed predation affects selection on several plant traits, such as flowering phenology and flower number, which are usually interpreted mainly in the context of plant–pollinator interactions. (4) The patterns of variation in the interactions between plants and pre-dispersal seed predators suggest that geographic selection mosaics may be common. (5) Although there are numerous studies estimating seed predation, there are still rather few studies that have aimed at examining the interaction explicitly in terms of effects on plant population dynamics and trait selection. From these we know that seed predators can have important, and often variable, effects on plant population dynamics and trait evolution. However, it still remains to assess how important they are across study systems and relative to other aspects of the plant's biotic and abiotic environment.     

Mycorrhiza Symbiosis Increases the Surface for Sunlight Capture in Medicago truncatula for Better Photosynthetic Production

Arbuscular mycorrhizal (AM) fungi play a prominent role in plant nutrition by supplying mineral nutrients, particularly inorganic phosphate (P_i), and also constitute an important carbon sink. AM stimulates plant growth and development, but the underlying mechanisms are not well understood. In this study, Medicago truncatula plants were grown with Rhizophagus irregularis BEG141 inoculum (AM), mock inoculum (control) or with P_i fertilization. We hypothesized that AM stimulates plant growth through either modifications of leaf anatomy or photosynthetic activity per leaf area. We investigated whether these effects are shared with P_i fertilization, and also assessed the relationship between levels of AM colonization and these effects. We found that increased P_i supply by either mycorrhization or fertilization led to improved shoot growth associated with increased nitrogen uptake and carbon assimilation. Both mycorrhized and P_i-fertilized plants had more and longer branches with larger and thicker leaves than the control plants, resulting in an increased photosynthetically active area. AM-specific effects were earlier appearance of the first growth axes and increased number of chloroplasts per cell section, since they were not induced by P_i fertilization. Photosynthetic activity per leaf area remained the same regardless of type of treatment. In conclusion, the increase in growth of mycorrhized and P_i-fertilized Medicago truncatula plants is linked to an increase in the surface for sunlight capture, hence increasing their photosynthetic production, rather than to an increase in the photosynthetic activity per leaf area.     

PACAP enhances the expression of CD11b,CD66b and CD63 in human neutrophils

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.     

Warsaw Breakage Syndrome, a Cohesinopathy Associated with Mutations in the XPD Helicase Family Member DDX11/ChlR1

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.     

Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides

Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an "S"-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.