Inducible Promoter Research Articles

Real-time detection of response regulator phosphorylation dynamics in live bacteria

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct invivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.

A universal approach to gene expression engineering.

In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5ʹ regulatory sequences (ARES) consisting of promoters and 5ʹ untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus and Streptomyces lividans, and the eukaryote yeast Saccharomyces cerevisiae.

Heterologous expression of C30 carotenoid biosynthetic gene crtNM from Lactiplantibacillus plantarum.

Probiotic lactic acid bacterium Lactiplantibacillus plantarum is widely used in the dairy and other fermented food industries. L. plantarum AR113 harbors a C30 carotenoid operon crtNM based on genomic analysis, but the yield of C30 carotenoid is only 8.1μg g-1 DCW. To improve the productivity of C30 carotenoid, crtNM from L. plantarum AR113 was cloned and reconstructed in Escherichia coli BL21(DE3). The proteins crtN and crtM were successfully expressed based on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the carotenoid was detected using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In comparison with the constitutive promoter P44 , the use of the inducible T7 promoter significantly increased the carotenoid content in E. coli. The fermentation conditions were also optimized with induction by 0.5 mmol/L IPTG at 20 °C for 7h. The yield of C30 carotenoid reached 154.5μg g-1 DCW, which was 18-fold higher than that of L. plantarum AR113. The 2,2-diphenyl-1-picryl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid (ABTS) radical scavenging capacity of C30 carotenoids synthesized by heterologous expression in E. coli was also higher than that of the antioxidant food additive butylated hydroxytoluene. Our findings suggest that E. coli has strong potential as a basic chassis for the production of C30 carotenoids from Lactiplantibacillus with high antioxidant activity. © 2022 Society of Chemical Industry.

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

The Sclerotinia sclerotiorum-inducible promoter pBnGH17D7 in Brassica napus: isolation, characterization, and application in host-induced gene silencing.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is among the most devastating diseases in Brassica napus worldwide. Conventional breeding for SSR resistance in Brassica species is challenging due to the limited availability of resistant germplasm. Therefore, genetic engineering is an attractive approach for developing SSR-resistant Brassica crops. Compared with the constitutive promoter, an S. sclerotiorum-inducible promoter would avoid ectopic expression of defense genes that may cause plant growth deficits. In this study, we generated a S. sclerotiorum-inducible promoter. pBnGH17D7, from the promoter of B. napus glycosyl hydrolase 17 gene (pBnGH17). Specifically, 5'-deletion and promoter activity analyses in transgenic Arabidopsis thaliana plants defined a 189 bp region of pBnGH17 which was indispensable for S. sclerotiorum-induced response. Compared with pBnGH17, pBnGH17D7 showed a similar response upon S. sclerotiorum infection, but lower activity in plant tissues in the absence of S. sclerotiorum infection. Moreover, we revealed that the transcription factor BnTGA7 directly binds to the TGACG motif in pBnGH17D7 to activate BnGH17. Ultimately, pBnGH17D7 was exploited for engineering Sclerotinia-resistant B. napus via host-induced gene silencing. It induces high expression of siRNAs against the S. sclerotiorum pathogenic factor gene specifically during infection, leading to increased resistance.

Production of Functional Recombinant Human Interferon-gamma by RTX CPD-fusion Technology

Human interferon-gamma (hIFNγ) is an important pleiotropic cytokine with therapeutic application. Recombinant hIFNγ has been shown to be an effective pharmaceutical against a wide range of viral, immuno-suppressive diseases with promising prospects in cancer immunotherapy resulting in a strong increase in demand and price. The aim of this study was to develop an efficient method for production of soluble and active hIFNγ. To this end a fusion gene encoding hIFNγ and a C-terminally located His10-tagged RTX CPD was constructed by two-step PCR. The fusion gene was cloned in an expression vector under inducible promoter and expressed in different E. coli strains such as Rosetta (appropriate for expression of eukaryotic genes containing rare codons), and two chaperone containing strains – BL21(DE3)/pG-KJE8 and BL21(DE3)/pG-Tf2. The transformed bacteria were cultivated at 24℃ to favour proper folding of the recombinant protein. Under these conditions the strain BL21(DE3)/pG-KJE8 was chosen as more suitable for production of soluble hIFNγ. The latter was purified by Ni2+- based affinity chromatography, the peak fractions were subjected to affinity chromatography combined with a direct on-column digestion to release hIFNγ, which was further purified by ion exchange chromatography. It is worth mentioning that this strategy, employing auto-cleaving and solubility-enhancing CPD tag, combined with on-column tag digestion was applied for the first time for production of hIFNγ.

A Model for Pharmaceutical Proteins Production from Arabidopsis Suspension Culture

Pharmaceutical proteins production in plant suspension culture has been of interest due to the ease of manipulation, low cost and low allergenic reaction in humans. In this work, the constitutive double CaMV 35S promoter and the abiotic stress-responsive promoter of the Rd29a locus were compared to produce Green Fluorescent Protein (GFP) used as a model for secreted protein in Arabidopsis thaliana suspension culture. Induction of Rd29a inducible promoter by plant hormone abscisic acid (ABA) was also studied. A signal sequence (SS) of a 33 kDa rice protein was added to the constructs to promote secretion. The secretion of GFP into the extracellular spaces of the transgenic seedling was detected under confocal microscope. The fl uorescence intensity of GFP in the suspension culture was measured by fl uorometry. A temporal maximum expression of GFP was observed from the CaMV 35S promoter on day 18. This model can be used as an example to produce pharmaceutical proteins in suspension culture in the future.

GAS6/TAM signaling pathway controls MICA expression in multiple myeloma cells.

NKG2D ligands play a relevant role in Natural Killer (NK) cell -mediated immune surveillance of multiple myeloma (MM). Different levels of regulation control the expression of these molecules at cell surface. A number of oncogenic proteins and miRNAs act as negative regulators of NKG2D ligand transcription and translation, but the molecular mechanisms sustaining their basal expression in MM cells remain poorly understood. Here, we evaluated the role of the growth arrest specific 6 (GAS6)/TAM signaling pathway in the regulation of NKG2D ligand expression and MM recognition by NK cells. Our data showed that GAS6 as well as MERTK and AXL depletion in MM cells results in MICA downregulation and inhibition of NKG2D-mediated NK cell degranulation. Noteworthy, GAS6 derived from bone marrow stromal cells (BMSCs) also increases MICA expression at both protein and mRNA level in human MM cell lines and in primary malignant plasma cells. NF-kB activation is required for these regulatory mechanisms since deletion of a site responsive for this transcription factor compromises the induction of mica promoter by BMSCs. Accordingly, knockdown of GAS6 reduces the capability of BMSCs to activate NF-kB pathway as well as to enhance MICA expression in MM cells. Taken together, these results shed light on molecular mechanism underlying NKG2D ligand regulation and identify GAS6 protein as a novel autocrine and paracrine regulator of basal expression of MICA in human MM cells.

Cohesin couples transcriptional bursting probabilities of inducible enhancers and promoters

Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways.

RhgKL and CcpA directly regulate the rhiLFGN-rhgR-yesTUV operon involved in the metabolism of rhamnogalacturonan type I in Bacillus subtilis.

The Bacillus subtilis rhiLFGN-rhgR-yesTUVWXYZ (formerly yesOPQRSTUVWXYZ) gene cluster includes genes for metabolizing rhamnogalacturonan type I (RG-I), a major pectin constituent, and the rhgR gene encoding an AraC/XylS transcriptional activator. The yesL-rhgKL (formerly yesLMN) operon, adjacent to the rhiL gene, includes the rhgKL genes encoding a two-component regulatory system. The reporter analyses showed that 3 promoters immediately upstream of the rhiL, yesW, and yesL genes were induced by RG-I and repressed by glucose in the medium. The reporter analyses also showed that RhgL and RhgR contribute to the RG-I-dependent induction of the rhiL promoter and that CcpA mediates the catabolite repression of the rhiL and yesL promoters. The in vitro experiments demonstrated that the RhgL response regulator and the CcpA complex bind to each site in the rhiL promoter region. The RT-PCR analysis and the different properties of the rhiL and yesW promoters suggested the rhiLFGN-rhgR-yesTUV genes as an operon.

Regulated Expression of lpxC Allows for Reduction of Endotoxicity in Bordetella pertussis.

The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria.

A Novel Transcriptional Regulator HbERF6 Regulates the HbCIPK2-Coordinated Pathway Conferring Salt Tolerance in Halophytic Hordeum brevisubulatum.

Halophytic Hordeum brevisubulatum is a perennial grass which has evolved many distinctive salt-adaptive mechanisms. Our previous studies indicated it could thrive under salt stress through maintaining better K+ and Na+ homeostasis. Stress-responsive HbCIPK2 can phosphorylate K+ channel HbVGKC1 and Na+ transporter HbSOS1L to prevent Na+ accumulation and K+ reduction, hence pathway was not detected in glycophytic plants. In this study, we cloned the inducible promoter of HbCIPK2 by genome-walking, and identified a novel transcriptional regulator HbERF6 through yeast one-hybrid screening. HbERF6 functioned as a transcription factor which can bind to the GCC-box of the HbCIPK2 promoter to activate its expression. HbERF6 transgenic lines in Arabidopsis improved salt tolerance compared with wild type, and especially induced AtCIPK24 (SOS2) expression, resulting in K+/Na+ homeostasis to enhance salt tolerance. All the results confirmed the inducible function of HbERF6 for CIPK genes during salt tolerance. This regulatory network that integrates transcriptional regulation and post-translation modification will unravel a novel salt stress-responsive mechanism, highlighting the value and utilization of the halophytic resource.

Efficient Robust Yield Method for Preparing Bacterial Ghosts by Escherichia coli Phage ID52 Lysis Protein E.

Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production, E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD600 reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose.

Enhanced protein translocation to mammalian cells by expression of EtgA transglycosylase in a synthetic injector E. coli strain

BackgroundBacterial type III secretion systems (T3SSs) assemble a multiprotein complex termed the injectisome, which acts as a molecular syringe for translocation of specific effector proteins into the cytoplasm of host cells. The use of injectisomes for delivery of therapeutic proteins into mammalian cells is attractive for biomedical applications. With that aim, we previously generated a non-pathogenic Escherichia coli strain, called Synthetic Injector E. coli (SIEC), which assembles functional injectisomes from enteropathogenic E. coli (EPEC). The assembly of injectisomes in EPEC is assisted by the lytic transglycosylase EtgA, which degrades the peptidoglycan layer. As SIEC lacks EtgA, we investigated whether expression of this transglycosylase enhances the protein translocation capacity of the engineered bacterium.ResultsThe etgA gene from EPEC was integrated into the SIEC chromosome under the control of the inducible tac promoter, generating the strain SIEC-eEtgA. The controlled expression of EtgA had no effect on the growth or viability of bacteria. Upon induction, injectisome assembly was ~ 30% greater in SIEC-eEtgA than in the parental strain, as determined by the level of T3SS translocon proteins, the hemolytic activity of the bacterial strain, and the impairment in flagellar motility. The functionality of SIEC-eEtgA injectisomes was evaluated in a derivative strain carrying a synthetic operon (eLEE5), which was capable of delivering Tir effector protein into the cytoplasm of HeLa cells triggering F-actin polymerization beneath the attached bacterium. Lastly, using β-lactamase as a reporter of T3SS-protein injection, we determined that the protein translocation capacity was ~ 65% higher in the SIEC-EtgA strain than in the parental SIEC strain.ConclusionsWe demonstrate that EtgA enhances the assembly of functional injectisomes in a synthetic injector E. coli strain, enabling the translocation of greater amounts of proteins into the cytoplasm of mammalian cells. Accordingly, EtgA expression may boost the protein translocation of SIEC strains programmed as living biotherapeutics.

Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.

Implementation of a tunable t-CRISPRi system for gene regulation in Giardia duodenalis

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.

NbNAC42 and NbZFP3 Transcription Factors Regulate the Virus Inducible NbAGO5 Promoter in Nicotiana benthamiana.

Plant argonautes (AGOs) play important roles in the defense responses against viruses. The expression of Nicotiana benthamiana AGO5 gene (NbAGO5) is highly induced by Bamboo mosaic virus (BaMV) infection; however, the underlying mechanisms remain elusive. In this study, we have analyzed the potential promoter activities of NbAGO5 and its interactions with viral proteins by using a 2,000 bp fragment, designated as PN1, upstream to the translation initiation of NbAGO5. PN1 and seven serial 5′-deletion mutants (PN2–PN8) were fused with a β-glucuronidase (GUS) reporter and introduced into the N. benthamiana genome by Agrobacterium-mediated transformation for further characterization. It was found that PN4-GUS transgenic plants were able to drive strong GUS expression in the whole plant. In the virus infection tests, the GUS activity was strongly induced in PN4-GUS transgenic plants after being challenged with potexviruses. Infiltration of the transgenic plants individually with BaMV coat protein (CP) or triple gene block protein 1 (TGBp1) revealed that only TGBp1 was crucial for inducing the NbAGO5 promoter. To identify the factors responsible for controlling the activity of the NbAGO5 promoter, we employed yeast one-hybrid screening on a transcription factor cDNA library. The result showed that NbNAC42 and NbZFP3 could directly bind the 704 bp promoter regions of NbAGO5. By using overexpressing and virus-induced gene silencing techniques, we found that NbNAC42 and NbZFP3 regulated and downregulated, respectively, the expression of the NbAGO5 gene. Upon virus infection, NbNAC42 played an important role in regulating the expression of NbAGO5. Together, these results provide new insights into the modulation of the defense mechanism of N. benthamiana against viruses. This virus inducible promoter could be an ideal candidate to drive the target gene expression that could improve the anti-virus abilities of crops in the future.

Constitutive Active CPK30 Interferes With Root Growth and Endomembrane Trafficking in Arabidopsis thaliana.

Calcium-dependent protein kinases (CPK) are key components of a wide array of signaling pathways, translating stress and nutrient signaling into the modulation of cellular processes such as ion transport and transcription. However, not much is known about CPKs in endomembrane trafficking. Here, we screened for CPKs that impact on root growth and gravitropism, by overexpressing constitutively active forms of CPKs under the control of an inducible promoter in Arabidopsis thaliana. We found that inducible overexpression of an constitutive active CPK30 (CA-CPK30) resulted in a loss of root gravitropism and ectopic auxin accumulation in the root tip. Immunolocalization revealed that CA-CPK30 roots have reduced PIN protein levels, PIN1 polarity defects and impaired Brefeldin A (BFA)-sensitive trafficking. Moreover, FM4-64 uptake was reduced, indicative of a defect in endocytosis. The effects on BFA-sensitive trafficking were not specific to PINs, as BFA could not induce aggregation of ARF1- and CHC-labeled endosomes in CA-CPK30. Interestingly, the interference with BFA-body formation, could be reverted by increasing the extracellular pH, indicating a pH-dependence of this CA-CPK30 effect. Altogether, our data reveal an important role for CPK30 in root growth regulation and endomembrane trafficking in Arabidopsis thaliana.

Abstract 558: Focused IL-12 cytokine delivery enhances function of affinity-tuned and real-time tracked ICAM-1-specific CAR T cells in solid tumors

Abstract Engineering CAR T cells to target solid tumors presents numerous challenges. Lack of tumor-specific antigens, high probability of CAR T cell exhaustion, and an often-immunosuppressive tumor microenvironment limit CAR T cell efficacy and outcomes in the clinic. To address these challenges, AffyImmune has developed “Tune & Track” technologies to reduce CAR affinity for antigens with disproportionally high density on tumor cells. Our CAR T cells are selective for tumor cells while leaving normal cells untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. CAR T cells are also designed to express Somatostatin Receptor 2 (SSTR2), which allows for tracking in vivo via PET/CT scan using FDA-approved DOTATATE. Our primary CAR design called AIC100 expresses an affinity-tuned variant of LFA-1, which is the natural ligand for Intracellular Adhesion Molecule (ICAM)-1, a molecule that is highly expressed on some cancers. A single dose of AIC100 CAR T cells results in elimination of tumors in mice and significantly improves survival. We have tested the safety and anti-tumor activity of AIC100 in high ICAM-1-expressing animal tumor models including triple-negative breast cancer and gastric cancer. AffyImmune is currently conducting a Phase I study (NCT04420754) to evaluate the clinical efficacy of AIC100 in treating advanced thyroid cancer. After receiving 10 million AIC100 CAR T cells, FDG and DOTATATE imaging revealed preliminary evidence of CAR T expansion in the patient and tumor reduction at multiple lesions. To further optimize affinity-tuned CAR T cell activity within tumors, we are currently developing CAR designs that have the capability to deliver effector cytokines to the tumor microenvironment via a nuclear factor of activated T cells (NFAT)-inducible promoter. Because our CAR T cells are affinity-tuned, off-tumor cytokine secretion is minimized. Our ICAM-1-specific CAR design called AIC1010 releases interleukin (IL)-12 cytokine upon engagement with target antigen within tumors, which induces production of interferon (IFN)-γ from immune cells and surrounding tissues, enhancing not only CAR T cell effector function but also the immune response focused within the tumor microenvironment. AIC1010 CAR T cells demonstrate superior cancer elimination compared to AIC100 CAR T cells in subcutaneous and IP tumor models in mice. We anticipate that cytokine delivery, paired with our “Tune & Track” technology will be widely adaptable to develop potent and safe affinity-tuned CAR T cells against hard-to-treat solid cancers. Citation Format: Jingmei Hsu, Michael P. Gallagher, Yanping Yang, Eric von Hofe, Yen-Michael Hsu, Koen Van Besien, Thomas Fahey, Jana Ivanidze, Janusz Puc, Karrie Du, Moonsoo Jin. Focused IL-12 cytokine delivery enhances function of affinity-tuned and real-time tracked ICAM-1-specific CAR T cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 558.

Sphingolipid Long-Chain Base Phosphate Degradation Can Be a Rate-Limiting Step in Long-Chain Base Homeostasis.

Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels.