Functional Reconstitution Research Articles

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Structure and Reconstitution of an MCU–EMRE Mitochondrial Ca2+ Uniporter Complex

The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU–EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Using a novel assay, we demonstrate robust Ca2+ uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU–EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.

Molecular Crux of Hair Cell Mechanotransduction Machinery

Although 62 years have elapsed since the first report of hereditary deafness in a mouse strain, the molecular mechanism of hair cell mechanotransduction remains elusive. Three recent studies present crucial insights into the molecular crux of hair cell mechanotransduction machinery.

Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases.

The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

Evaluation of Functional Characteristics and Reconstitution Behavior of Formulated Ready to Reconstitute Enteral Formula for Nutrition Therapy

Aims: The present investigation was undertaken to evaluate the functional properties and reconstitution behavior of enteral formulae i.e. Balanced Enteral Formula, High Protein Enteral Formula and High Energy Enteral Formula formulated using locally available natural ingredients.
 Place and Duration of Study: The study was conducted in the Department of Food Science and Nutrition, College of Community Science, Assam Agricultural University during the period of August 2017 to January 2018. 
 Methodology: Three enteral formulae namely Balanced Enteral Formula, High Protein Enteral Formula and High Energy Enteral Formula were formulated using naturally available low cost ingredients. Standard protocols were followed to evaluate the quality of ingredients used and formulated enteral formulae. 
 Results: Both ingredients and enteral formulae had good functional properties with bulk density, tapped density, particle density, porosity, cohesiveness, flowability, water absorption index and water solubility index ranging from 0.33 to 0.50 g/ml, 0.38 to 0.56 g/ml, 0.59 to 1.25 g/ml, 35.00 to 60.00per cent, 0.33 to 1.18, 0.00 to 17.77 per cent, 1.23 to 7.59 g/g and 23.66 to 87.62 per cent soluble solids respectively for the ingredients used and 0.44 to 0.47 g/ml; 0.50 to 0.53 g/ml; 0.89 to 0.90 g/ml; 41.00 to 44.44 per cent; 1.12 to 1.14; 11.30 to 12.00 per cent; 3.39 to 4.59 g/g and 42.66 to 51.00 per cent respectively for the formulated enteral formulae. Color analysis of the developed formulae using Hunter Lab showed desirable results with L*, a* and b* value in the range of 82.43 to 87.10; 1.35 to 2.38 and 13.75 to 13.95 respectively. The reconstitution of formulated enteral formulae with warm water revealed a reconstitution time varying from 5 to 6 minutes. Further the reconstituted formula feed on passage through Ryles tube showed superior flow rate in formulae prepared with 20 per cent solid concentration (W/V).
 Conclusion: The developed enteral formulae had excellent functional characteristics and reconstitution behavior indicating quality product suitable for enteral feeding.

A novel approach for dynamic in-situ surface characterisation of milk protein concentrate hydration and reconstitution using an environmental scanning electron microscope

Composition and relative humidity (RH) can have a profound impact on the physical (flowability, stickiness) and functional (reconstitution) properties of milk powder (MP) and therefore its quality, storage stability and shelf-life. Conventional microscopic techniques are not capable of dynamically imaging the effect of RH on MP at high magnification. The aim of this study was to develop a novel method to characterise in-situ and in real time the hydration and reconstitution of five spray-dried milk protein concentrates (MPCs) using an Environmental Scanning Electron Microscope (ESEM). ESEM was employed to observe the surface microstructure of MPC powders with varying protein content (38.63%–80.94%, w/w), at various RH values ranging from 35% to over 100%. MPC powders were imaged by an ESEM without any prior preparation, and with minimal physical sample alteration, thus providing fundamental insights into MPC hydration and reconstitution. ESEM surface analysis showed particle swelling in all MPCs, and that with increasing protein content, hydration and reconstitution efficiency decreased. For the first time, dynamic particle surface fusion was observed. Such fusion can result in stickiness and caking over time. ESEM methods developed here may provide mechanistic insights into the effects of RH during storage. Surface re-arrangement was also observed in all MPCs, but was impeded in MPC70 and MPC80 thus indicating that this is the rate limiting step for MPC reconstitution. This work validates the use of an ESEM to dynamically characterise MPC powder hydration and reconstitution in-situ and in real-time, at both high magnification and spatial resolution.

Photosynthetic Model Membranes of Natural Plant Thylakoid Embedded in a Patterned Polymeric Lipid Bilayer.

Thylakoid membranes in the chloroplast of plants, algae, and cyanobacteria are the powerhouse of photosynthesis, capturing solar energy and converting it into chemical energy. Although their structures and functions have been extensively studied, the intrinsically heterogeneous and dynamic nature of the membrane structures is still not fully understood. Investigating native thylakoid membranes in vivo is difficult due to their small size and limited external access to the chloroplast interior, while the bottom-up approaches based on model systems have been hampered by the sheer complexity of the native membrane. Here, we try to fill the gap by reconstituting the whole thylakoid membrane into a patterned substrate-supported planer bilayer. A mixture of thylakoid membrane purified from spinach leaves and synthetic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles spontaneously formed a laterally continuous and fluid two-dimensional (2D) membrane in the scaffold of the patterned polymeric bilayer. Chlorophyll fluorescence arising from photosystem II (PSII) recovered after photobleaching, suggesting that the membrane components are laterally mobile. The reversible changes of chlorophyll fluorescence in the presence of the electron acceptors and/or inhibitors indicated that the electron transfer activity of PSII was retained. Furthermore, we confirmed the electron transfer activity of photosystem I (PSI) by observing the generation of nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of water-soluble ferredoxin and ferredoxin-NADP+ reductase. The lateral mobility of membrane-bound molecules and the functional reconstitution of major photosystems provide evidence that our hybrid thylakoid membranes could be an excellent experimental platform to study the 2D molecular organization and machinery of photosynthesis.

IroT/MavN is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by In‐vitro Real‐time Transport

Iron plays a vital role in all living organisms due to its key role as an enzyme co‐factor, central to diverse metabolic processes. However, because of its high reactivity, speciation, and insolubility in oxidative environments, organisms have evolved sophisticated networks for intercellular iron acquisition without reaching toxic levels. In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, the causative agent of Legionnaires’ disease, a putative transmembrane protein, IroT/MavN, is inserted onto the surface of the host pathogen‐containing vacuole to facilitate intravacuolar iron acquisition from the host, bypassing the problem of Fe(III) insolubility and mobilization.In this work, we have developed a platform for purification and the functional reconstitution of IroT/MavN in artificial lipid bilayer vesicles (proteoliposomes) to identify and characterize the IroT/MavN mediated metal transport properties. By encapsulating the fluorescent reporter probe Fluozin‐3, by real‐time metal transport assays, we revealed that IroT/MavN is a high‐affinity and high‐capacity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains and loops important for substrate recognition and rapid‐kinetic translocation. In addition, by encapsulating the pH indicator pyranine, we demonstrated that Fe(II) translocation is coupled to H+ counter‐transport, suggesting that IroT/MavN is a Fe(II)‐H+ antiporter.This work establishes the substrate transport properties involved in a novel transporter family important for iron acquisition at the host‐pathogen intravacuolar interface and provides chemical tools for comparative investigation of the translocation properties in other iron transporter families.Support or Funding InformationThe work was supported by the Robert A. Welch Foundation (AT‐1935‐20170325 to G.M.) and by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM128704 to G.M.).

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1−/− mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34+ cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.

Mixed hybrid bilayer lipid membranes on mechanically polished titanium surface

Mixed self-assembled monolayers of octadecyltrichlorosilane (OTS) and methyltrichlorosilane (MTS) were deposited via simple silanization procedure on a mechanically polished titanium surface. The monolayers act as molecular anchors for mixed hybrid bilayer lipid membranes (mhBLM) which were accomplished via vesicle fusion. A variation of the MTS concentration in silanization solutions significantly affects properties of mhBLMs composed of a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol). The bilayers become less insulating following an increase of the MTS content. On the other hand, an increase of the MTS concentration provides flexibility of the mhBLM membranes necessary for the functional reconstitution of membrane proteins. The optimal molar ratio of MTS in silanization solution is 40% providing anchors for intact mhBLMs as confirmed by their specific capacitance of 0.86 μF cm−2. We found that the bilayers containing 40% (mol) of cholesterol bind cholesterol dependent pneumolysin (PLY). However, we did not observe functional reconstitution of PLY. While α-hemolysin almost fully disrupts mhBLMs assembled from 100% diphytanoyl. An important advantage of the titanium/OTS/MTS molecular anchor systems is their ability of repetitive regeneration of phospholipid bilayers without losing functional properties as demonstrated in the current study. This creates a possibility for the multiple-use phospholipid membrane biosensors which have a potential of decreasing the cost of such electrochemical/electroanalytical devices.

Cell-Free Expression Systems: Probing Nuclear Mechanotransduction Using Novel Engineering Platforms

Cell-free expression (CFE) of proteins has become a versatile tool for in vitro reconstitution studies. Among the many advantages of using such systems, functional reconstitution of membrane proteins is a major improvement over traditional purification alternatives. In this work, we use a CFE system derived from HeLa cells to functionally reconstitute membrane proteins in artificial lipid bilayer membranes. SUN proteins are inner nuclear membrane proteins which have been shown to play a major role in nuclear mechanotransduction. SUN proteins bind to Nesprins that are known to interact with cytoskeletal filaments, via SUN-KASH (C-terminal peptide in Nesprins) binding which results in force transmission from the cytoskeleton to the nuclear lamina. While the assembly of SUN-KASH complex is known, the topology of SUN proteins in the inner nuclear membrane and their range of interactions with other nuclear membrane proteins remain unexplored due to several challenges with traditional cell biology approaches. Here, we expressed SUN proteins using our CFE system on lipid bilayer-coated beads in vitro as a platform for studying topology of membrane protein reconstitution. We present compelling evidence to confirm ER microsomes-mediated membrane protein reconstitution and show the versatility of our approach to probe the interaction of SUN proteins and their mutants with the membrane and investigate their orientation of insertion in the bilayer. Further we demonstrate functionality of reconstituted SUN proteins by investigating their binding to KASH peptides and other SUN mutants. The results presented here indicate the possibility of using CFE systems as tools for probing protein functions in vitro.

Agonist Selectivity and Ion Permeation in the α3β4 Ganglionic Nicotinic Receptor

Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3β4 nicotinic receptor subtype forms the principal relay between the central and peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the α3β4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the α3β4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Functional Reconstitution of the 5-HT3 Receptor

The 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC) embedded in the membrane of neurons, which plays an important role during fast neurotransmission in the brain and the gut. Mutations in this receptor have been associated to neurological and psychiatric disorders such as epilepsy, depression and schizophrenia; as a result, 5-HT3 receptor, among other pLGICs, is the target of a legion of therapeutic compounds. Therefore, understanding the molecular mechanisms of the 5-HT3 receptor can provide both fundamental biological insights and potential benefits to health.Our laboratory has previously obtained several structures of the 5-HT3 receptor in different conformations: inhibited, active open-pore and pre-active using both crystallography and Cryo-EM. All these structures, that shed light on conformational transitions of the 5-HT3 receptor, have been obtained in the presence of detergent. Detergents are useful biochemical tools; however, they might distort protein structure. Their putative deleterious influence should be assessed on case-by-case basis. The aim of the project is to achieve the functional reconstitution of the 5-HT3 receptor by evaluating several membrane mimetics. The most interesting results so far have been obtained with saposin A nanoparticles, with which we could obtain cryo-EM datasets of reconstituted 5-HT3 receptor. Surprisingly, while the conformation of the extracellular domain is similar to that in detergent, the structure shows an important level of distortion in the transmembrane α-helices. We are now investigating more extensively how various receptor to lipids to saposin A ratios lead to transmembrane domain distortions.

Magnetic Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state nuclear magnetic resonance (NMR) spectroscopy and aids in the measurement of residual dipolar couplings via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic alignment properties of styrene maleimide quaternary ammonium (SMA-QA) polymer-based nanodiscs by using 31P and 14N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic alignment of large-sized (≥20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid bilayer normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs is further shown to flip the alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

Expression, purification, and functional reconstitution of 19F-labeled cytochrome b5 in peptide nanodiscs for NMR studies

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

Functional reconstitution of TatB into the thylakoidal Tat translocase

We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analysed with the authentic Tat substrate pOEC16, reached routinely 20–25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutated TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

Donor and Host Coexpressing KIR Ligands Promote NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation

Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded. Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells. Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR. Results: 1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2. 2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)). 3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness. 4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022). Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients. Disclosures No relevant conflicts of interest to declare.

Maintaining the Cellular Anti-Viral and Anti-Leukemic Activities in GvHD Patients Undergoing Extracorporeal Photophoresis Therapy

Introduction: Systemic steroids with their strong immunosuppressive and anti-inflammatory effects are the current gold standard for initial treatment of the graft-versus-host disease (GvHD). But, the effectiveness of high-dose steroids comes along with an increased susceptibility to infections and other secondary malignancies. Extracorporeal photophoresis (ECP), an immunotherapeutic therapy, is currently being used as a clinically well-established second-line treatment for GvHD. Contrary to steroid treatment, there is no clinical data showing that ECP is associated with an increased risk of infection. However, the exact mechanism of action how ECP preserves the anti-viral and anti-leukemic effects on the cellular level has not been discovered yet. Materials and methods: Thirty-four patients with steroid-refractory/resistant aGvHD ≥ II and moderate to severe cGvHD were treated with ECP at the University Hospitals in Heidelberg, Greifswald in Germany and Chaim Sheba Medical Center in Israel. A comprehensive analysis of cell subsets was performed using multi-parametric flow cytometry. Additionally, the quantity and quality of CMV-specific T cells was determined by tetramer staining and interferon-γ enzyme-linked immunospot assay. The NK activity in terms of killing functionality and cytokine release was analyzed by intracellular cytokine staining and chromium-51 release assay, respectively. The proliferative capacity of effector cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Results: ECP therapy was effective with an overall response rate of 75% for patients with aGvHD (x/y) and 78% (x/y) for patients with cGvHD. Of note, all patients showed neither the increased susceptibility to infections nor the reactivation of CMV nor the tumor relapse. Overall, the frequency of well-established protective cell subsets in terms of cytotoxic CD8+ T cells, CD4+CD8+ T cells, γδ T cells, NK cells and NKT cells remained constant under the ECP therapy. Specifically, no significant influence of ECP therapy on the CD56dimCD57+NKG2C+ NK cells, a specialized anti-viral/tumor population, and the CMV+CD8+ T cells could be observed. The further phenotyping of CMV+CD8+ T cells showed that CD45RA+CCR7-CMV+CD8+ TE cells and CD45RA-CCR7-CMV+CD8+ TEM cells could keep stable under the ECP therapy. Besides these, priming, differentiation and maturation of NK cells through upregulation of CD57 by ECP therapy bridged the T-cell-deficient period after HSCT in order to control the viral infections and eliminate the residual malignant cells. Moreover, ECP could reduce the CD62L expression on TE population which not only facilitates the hematopoietic engraftment and contributes to the phenotypic and functional T cell reconstitution after transplantation without causing GvHD, but also enhances the functional immune reconstitution against tumor and viral antigens. Functionally, neither IFN-g released by virus specific T cells nor production of TNF-a and IFN-g by NK cells in response to K562 cells was hampered by ECP. Of note, the functions of NK cells in terms of the MFI of cytokines and the polyfunctionality of NK cells were stable under ECP treatment. Additionally, the proliferation of NK cells, CD4+ T cells and CD8+ T cells providing an expanded pool of effector cells against the pathogens was not hampered by ECP therapy as well. Conclusions: ECP therapy constitutes an effective strategy to treat GvHD. In addition, ECP appears to be a safe therapy compared to continued steroid use. Our current results suggest that ECP allows to maintain immunity against infections and tumors. Disclosures Schönland: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Dreger:Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support .

Lentiviral Gene Therapy with Low Dose Busulfan for Infants with X-SCID Results in the Development of a Functional Normal Immune System: Interim Results of an Ongoing Phase I/II Clinical Study

Lentiviral Gene Therapy with Low Dose Busulfan for Infants with X-SCID Results in the Development of a Functional Normal Immune System: Interim Results of an Ongoing Phase I/II Clinical Study