Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies ( E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.

Although perilipin 2 (Plin2) has been shown to bind lipids with high affinity, the Plin2 lipid binding site has yet to be defined. This is of interest since Plin2’s affinity for lipids has been suggested to be important for lipid droplet biogenesis and intracellular triacylglycerol accumulation. To define these regions, mouse Plin2 and several deletion mutants expressed as recombinant proteins and in mammalian cells were assessed by molecular modeling, fluorescence binding, circular dichroic, and fluorescence resonance energy transfer techniques to identify the structural and functional requirements for lipid binding. Major findings of this study indicate (1) the N-terminal PAT domain does not bind cholesterol or stearic acid; (2) Plin2 residues 119–251, containing helix α4, the α–β domain, and part of helix α6 form a Plin3-like cleft found to be important for highest affinity lipid binding; (3) both stearic acid and cholesterol interact favorably with the Plin2 cleft formed by conserved residues in helix α6 and adjacent strands, which is common to all the active lipid-binding constructs; and (4) discrete targeting of the Plin2 mutants to lipid droplets supports Plin2 containing two independent, nonoverlapping lipid droplet targeting domains in its central and C-terminal sequences. Thus, the current work reveals specific domains responsible for Plin2–lipid interactions that involves the protein’s lipid binding and targeting functions.

e14263 Background: We have previously demonstrated that SPAR modified T cells effectively detect bacterial and viral pathogens. SPAR cells are engineered to express a modified T cell receptor (TCR) capable of antibody-directed signal transduction and respond with a Ca2+-mediated production of luminescent signal upon target detection. Here we describe the ability of SPAR cells to rapidly detect the cell surface expression of melanoma biomarkers without a requirement for extensive sample preparation. Methods: SPAR cells expressing both an engineered T cell receptor and the luminescent reporter protein aequorin were developed via the transfection of Jurkat cells with the aequorin expression vector pEF1-Aeq and an engineered TCR complex composed of mouse FcγRI fused to the CD3ζ subunit. The mFcγRI-CD3ζ receptor binds to the Fc region of full-length mouse IgG2 antibodies: SPAR cells are programmed for target detection via the addition of target-specific antibody. The ability of programmed SPAR cells to accurately detect melanoma-specific cell surface biomarkers was evaluated using whole cells from cultured mouse (B16-F10) and human (SK-Mel-28) melanoma cell lines. SPAR cells were programmed by incubation with murine antibodies directed toward the melanoma biomarkers CD133 or TRP1, then mixed with melanoma cells or K562 control cells and evaluated for signal generation. Results: SPAR cells programmed with anti-CD133 or anti-TRP1 antibody produced luminescent signal within minutes when combined with human SK-Mel-28 cells or mouse B16-F10 cells, respectively, known to abundantly express the appropriate biomarker. No signal was generated when programmed SPAR cells were incubated with K562 cells. Further studies also document cytokine production following receptor engagement. Conclusions: SPAR cells can be programmed for the rapid and specific detection of known cell surface cancer biomarkers. The Ca2+-dependent production of luminescent signal and cytokine release in response to TCR engagement suggests SPAR cell activation. Thus, in addition to biomarker detection the SPAR system may ultimately provide predictive insights into the potency of antibody-directed cell therapy.

8Efficient pathogen detection is essential for the successful treatment and prevention of infectious 9 disease; however, current methods are often too time intensive to be clinically relevant in cases 10 requiring immediate intervention. We have developed a Surface Programmable Activation 11 Receptor (SPAR) diagnostic platform comprised of universal biosensor cells engineered for use 12 in combination with custom or commercial antibodies to achieve rapid and sensitive pathogen 13 detection. SPAR cells are stably transfected Jurkat T cells designed to constitutively express a 14 modified T cell mouse FcγRI receptor on the cell surface and a high level of the luminescent 15 reporter protein aequorin in the cytoplasm. The modified mFcγRI-CD3ζ receptor protein binds 16 with high affinity to the Fc region of any full-length mouse IgG2a and some IgG2 antibodies: 17 this allows customized target detection via the selection of specific antibodies. T-cell receptor 18 aggregation in response to target antigen binding results in signal transduction which, when 19 amplified via the endogenous T cell signal cascade, triggers the rapid intracellular release of 20 calcium. Increased Ca 2+ concentrations activate the expressed reporter protein aequorin resulting 21 in the immediate emission of detectable light. Testing demonstrates the accurate and specific 22 2detection of numerous targets including P. aeruginosa, E. coli O111, and E. coli O157. We 23 report that the SPAR biosensor cell platform is a reliable pathogen detection method that enables 24 the rapid identification of bacterial causative agents using standard laboratory instrumentation. 25The technology lends itself to the development of efficient point-of-care testing and may aid in 26 the implementation of effective and pathogen-specific clinical therapies. 27 Introduction 28The rapid and accurate identification of causative agents is critical to the prompt application of 29 directed, pathogen-specific antibiotic therapies. Effective and timely clinical intervention is 30 essential for the control of infectious disease as well as in the successful treatment of bacterial 31 infections. The observed increases in the frequency and severity of nosocomial infections (1) 32 and the increasing prevalence of antibiotic resistance induced by non-specific antibiotic use (2) 33 further highlight the need for informed antibiotic selection based upon precise and efficient 34 pathogen detection. 35 Current bacterial identification methods include both classic procedures and novel molecular 36 techniques. Traditional culture-based methods, while sensitive and reliable, are also labor-37 intensive and time-consuming and therefore often cannot provide definitive diagnostics within a 38 clinically relevant timeframe (3). Molecular diagnostic methods include immunological assays 39 such as ELISA (4), microarray immunoblot (5), or serological assays (6); nucleic acid-based 40 techniques including PCR (7), DNA sequencing (8), hybridization techniques (9), or DNA/RNA 41 microarrays (10);...

Biomarkers play a central role in cancer management by facilitating detection and characterization and are key to the development of personalized therapeutic strategies. We have developed a novel Surface Programmable Activation Receptor (SPAR) diagnostic platform which uses T cell-based technology to achieve the rapid and sensitive identification of cancer biomarkers. SPAR cells are T cells modified to express both an engineered T-cell receptor (TCR) and the luminescent reporter aequorin. Jurkat T cells are first transfected with the aequorin expression vector pEF1-Aeq introduced via random insertion, and stable transfectants selected using G418. The TCR complex is functionally modified via the gene fusion of enhanced monoavidin (eMA) with the CD3ϵ subunit of the human T cell receptor complex to form eMA-CD3ϵ and introduced as a homozygous insert into Jurkat/pEF1-Aeq T cells via electroporation, using CRISPR/Cas9 targeting to replace endogenous CD3ϵ. The modified TCR complex efficiently binds biotinylated target detection molecules (TDMs), which target specific biomarkers via an engineered protein binding domain and a biotinylated protein tag, and act as a ligand to program the SPAR TCR. Simultaneous binding of the TDM to the target and to the modified TCR causes receptor aggregation. Aggregation signals are amplified via the native T-cell signal cascade and result in rapid calcium release from the endoplasmic reticulum. Increased calcium concentrations activate aequorin, triggering the emission of detectible light. Initially developed for programmable pathogen detection, the system has demonstrated reliable and specific detection of the lymphoma marker CD19. Cultured human Burkitt’s lymphoma (Raji B) cells abundantly expressing the CD19 surface receptor were combined with SPAR cells programmed with a biotinylated anti-CD19 antibody as TDM. Binding of the anti-CD19 SPAR cells to cell surface CD19 resulted in TCR aggregation, signal transduction, and the generation of reporter signal. Repeat studies conducted in the presence of blood demonstrated equivalent sensitivity. We achieve similar results using the human melanoma markers GD2 and CD133, and mouse melanoma markers TRP1 and CD44 in cell lines known to express these markers. In these studies, a variant of the SPAR system was used to screen commercial monoclonal antibodies to identify those with biologically relevant activity against the cell surface biomarkers, thus demonstrating the utility of this system in optimizing useful antibody/ target combinations without requiring target protein purification. With optimized TDMs, no significant light signal could be detected with K562 cells used as control. The SPAR diagnostic platform is a rapid and effective method for the detection of known cancer biomarkers and may provide a direct screening tool for the optimization of interacting proteins used to guide cell therapies. Citation Format: Joseph D. Kittle, Joel Lwande, M Russell Williams, Shengwen Liang, Kyle McQuaid, Melissa Frenchmeyer, Yuanyuan Tang, Allison Neese, Jiangzhou Hua, Charles McBrairty. Development of an engineered T cell receptor-based system for the rapid detection of cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3169.