Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Relationships of Lower Lung Fibrosis, Pleural Disease, and Lung Mass with Occupational, Household, Neighborhood, and Slate Roof-Dense Area Residential Asbestos Exposure.

This study aimed to evaluate the relationship between various asbestos exposure routes and asbestos-related disorders (ARDs). The study population comprised 11,186 residents of a metropolitan city who lived near asbestos factories, shipyards, or in slate roof-dense areas. ARDs were determined from chest X-rays indicating lower lung fibrosis (LFF), pleural disease (PD), and lung masses (LMs). Of the subjects, 11.2%, 10.4%, 67.2% and 8.3% were exposed to asbestos via occupational, household, neighborhood, and slate roof routes, respectively. The odds ratio (OR) of PD from household exposure (i.e., living with asbestos-producing workers) was 1.9 (95% confidence interval: 0.9⁻4.2), and those of LLF and PD from neighborhood exposure, or residing near asbestos factories) for <19 or >20 years, or near a mine, were 4.1 (2.8⁻5.8) and 4.8 (3.4⁻6.7), 8.3 (5.5⁻12.3) and 8.0 (5.5⁻11.6), and 4.8 (2.7⁻8.5) and 9.0 (5.6⁻14.4), respectively. The ORs of LLF, PD, and LM among those residing in slate-dense areas were 5.5 (3.3⁻9.0), 8.8 (5.6⁻13.8), and 20.5 (10.4⁻40.4), respectively. Substantial proportions of citizens residing in industrialized cities have potentially been exposed to asbestos, and various exposure routes are associated with the development of ARDs. Given the limitations of this study, including potential confounders such as socioeconomic status, further research is needed.

A Comparison between Low- and High-Passage Strains of Human Cytomegalovirus.

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log10 infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

The role of Arabidopsis ABCG9 and ABCG31 ATP binding cassette transporters in pollen fitness and the deposition of steryl glycosides on the pollen coat.

The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.

Sequencing and characterization of Varicella-zoster virus vaccine strain SuduVax.

BACKGROUND: Varicella-zoster virus (VZV) causes chickenpox in children and shingles in older people. Currently, live attenuated vaccines based on the Oka strain are available worldwide. In Korea, an attenuated VZV vaccine has been developed from a Korean isolate and has been commercially available since 1994. Despite this long history of use, the mechanism for the attenuation of the vaccine strain is still elusive. We attempted to understand the molecular basis of attenuation mechanism by full genome sequencing and comparative genomic analyses of the Korean vaccine strain SuduVax. RESULTS: SuduVax was found to contain a genome that was 124,759 bp and possessed 74 open reading frames (ORFs). SuduVax was genetically most close to Oka strains and these Korean-Japanese strains formed a strong clade in phylogenetic trees. SuduVax, similar to the Oka vaccine strains, underwent T- > C substitution at the stop codon of ORF0, resulting in a read-through mutation to code for an extended form of ORF0 protein. SuduVax also shared certain deletion and insertion mutations in ORFs 17, 29, 56 and 60 with Oka vaccine strains and some clinical strains. CONCLUSIONS: The Korean VZV vaccine strain SuduVax is genetically similar to the Oka vaccine strains. Further comparative genomic and bioinformatics analyses will help to elucidate the molecular basis of the attenuation of the VZV vaccine strains.

Full genome sequencing and analysis of human cytomegalovirus strain JHC isolated from a Korean patient.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Because of its huge size, comparative genomic studies of HCMV genome have been limited. In this study it was attempted to obtain and analyze the full genome sequence from clinical isolate from Korea. The strain JHC was isolated from Korean patient undergoing bone marrow transplantation who exhibited resistance to ganciclovir treatment (Lee et al., 2005). The virus was plaque-purified, and the full genome sequence was determined by pyrosequencing technique. The JHC genome was found to contain 235,476 bp and 165 open reading frames (ORFs). Comparison with the full genome nucleotide sequences of 11 other HCMV strains suggest that JHC is not closely related with any other strains at genome level. As expected, JHC lacked IRL sequences found in lab-adapted AD169-varUK strain and this region was replaced by ORFs UL133-UL150 as in other clinical isolates. Two ORFs (UL1 and UL119) of the strain JHC were found to be truncated due to early stop codons, and RL6 contains an unusual start codon TTG. The strain JHC contains all the genetic information for micro RNAs known to be present in HCMV.

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen.

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.

PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid.

Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants.

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis.

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1. The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae. The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

Rice P1B-type heavy-metal ATPase, OsHMA9, is a metal efflux protein.

P(1B)-type heavy-metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. Despite their importance, very little is known about their functions in monocot species. We report the characterization of rice (Oryza sativa) OsHMA9, a member of the P(1B)-type ATPase family. Semiquantitative reverse transcription-polymerase chain reaction analyses of seedlings showed that OsHMA9 expression was induced by a high concentration of copper (Cu), zinc (Zn), and cadmium. We also determined, through promoterbeta-glucuronidase analysis, that the main expression was in the vascular bundles and anthers. The OsHMA9:green fluorescence protein fusion was localized to the plasma membrane. Heterologous expression of OsHMA9 partially rescued the Cu sensitivity of the Escherichia coli copA mutant, which is defective in Cu-transporting ATPases. It did not rescue the Zn sensitivity of the zntA mutant, which is defective in Zn-transporting ATPase. To further elucidate the functional roles of OsHMA9, we isolated two independent null alleles, oshma9-1 and oshma9-2, from the T-DNA insertion population. Mutant plants exhibited the phenotype of increased sensitivity to elevated levels of Cu, Zn, and lead. These results support a role for OsHMA9 in Cu, Zn, and lead efflux from the cells. This article is the first report on the functional characterization of a P(1B)-type metal efflux transporter in monocots.