Development and implementation of natural killer cell simultaneous ADCC and direct killing assay

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing

Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)^(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF^(Met30) in vivo and in vitro, but not the closely related SCF^(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes

Method for VSV-G and Spike Viral Pseudotyping

Method for VSV-G and Spike Viral Pseudotyping Nathan Booher, Kelcy Swope, Paul W. Denton Ph.D. Department of Biology, University of Nebraska Omaha Introduction We are using a virus pseudotyping system to screen drugs that interfere with SARS-CoV-2 entry. To date, we have been focused on establishing the system in our laboratory. Methods We use a triple transfection approach in HEK-293T cells for pseudotyped virion production. Plasmid 1: The backbone virus for virion production is a Moloney Murine Leukemia Virus (MoMLV). Plasmid 2: This is a psi-element containing plasmid that encodes an EGFP reporter gene. Plasmid 3: This is a plasmid encoding the SARS-CoV-2 spike protein (variants will be introduced). Control Plasmid 3 encodes the envelope for Vesicular Stomatitis Virus G (VSVG). Viral particles are transduced onto VERO E6 cells which express the receptor for spike-mediated entry – ACE2. Transduction efficiency (proxy for viral entry) is quantitated via flow cytometry. Transfection was also monitored via fluorescence microscopy. Results, Discussion Initial results yielded a transduction efficiency of 3.4% with VSVG and lower with spike. This is not as high as we believe is possible and the result reveals that our pseudotyping method can be enhanced. Next steps include performing transductions in the presence of transduction enhancing agents (e.g polybrene). Other next steps include modulating plasmid rations during transfection to enhance virion production with maximal viral envelope incorporation. Once this system is functioning, we will work with our collaborators at UNMC to screen drugs with potential for interfering with SARS-CoV-2 entry into target cells

Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells

Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells Jaden Nienhueser1, Nathan Booher1, Paul W. Denton PhD1 1Department of Biology, University of Nebraska at Omaha Background: Human Immunodeficiency virus (HIV) affects nearly 40 million individuals worldwide. While antiretroviral therapy is effective against the virus, this therapy is a lifelong commitment. Thus, there is a strong global effort to develop interventions that are able to, at minimum, functionally cure this infection. Functional cure is when the body’s immune system is trained to control the HIV infection in the absence of ongoing antiretroviral therapy. Thus, multiple strategies are being investigated (e.g., immunotherapies in NCT03837756) to boost anti-HIV immunity. Our question is how such interventions may impact natural killer (NK) cell functions – as NK cells may be a critical component in controlling HIV in the absence of antiretroviral therapy. This project focuses on developing an ex vivo strategy for measuring the impact of relevant immunotherapies on human NK cell killing capacities with a particular focus on NK cell-mediated antibody dependent cellular cytotoxicity (ADCC). Methods: To accomplish our goals, we need a way to express HIV envelope on the surface of target cells. For this, we needed to logarithmically amplify the small plasmid stock provided by the NIH HIV Reagent Program. To do this, the HIV envelope gene containing (and ampicillin resistance cassette containing) plasmid Q842.D16 was transformed into competent Stbl2 Escherichia coli. Next, we grew transformed bacteria on LB agar plates supplemented with ampicillin. Following incubations, a colony of transformed bacteria was isolated from each plate. The colonies were amplified in LB broth supplemented with ampicillin. The most rapidly growing culture, based upon absorbance at 600 nm (Nanodrop 2000), was carried to the subsequent step of plasmid collection using a midi-prep kit (Zymo # D4200). Restriction enzyme fragmentation and subsequent gel electrophoresis was conducted to confirm the identity of the plasmid. Results: Our data confirmed that we amplified and purified the Q842.D16 plasmid. Our plasmid yield was ~660 ng/µL in ~3 mL, a quantity sufficient for long term use in the laboratory. Discussion: The overarching purpose of the processes completed for this abstract was to acquire an abundance of the Q842.D16 plasmid for transfecting potential target cells in our laboratory’s natural killer cell functional assay. We accomplished this goal. The next direction of the research is the focus of the partner poster being presented by Nathan Booher

Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing

Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing Nathan Booher1, Jaden Nienhueser1, Paul W. Denton PhD1 1Department of Biology, University of Nebraska at Omaha Introduction Human Immunodeficiency virus (HIV) affects nearly 40 million individuals worldwide. While antiretroviral therapy is effective against the virus, this therapy is a lifelong commitment. Thus, there is a strong global effort to develop interventions that are able to, at minimum, functionally cure this infection. Functional cure is when the body’s immune system is trained to control the HIV infection in the absence of ongoing antiretroviral therapy. Thus, multiple strategies are being investigated (e.g., immunotherapies in NCT03837756) to boost anti-HIV immunity. Our question is how such interventions may impact natural killer (NK) cell functions – as NK cells may be a critical component in controlling HIV in the absence of antiretroviral therapy. This project focuses on developing an ex vivo strategy for measuring the impact of relevant immunotherapies on human NK cell killing capacities with a particular focus on NK cell-mediated antibody dependent cellular cytotoxicity (ADCC). Methods To accomplish our goals, we need target cells for NK cells to kill. The target cells need to express HIV envelope that can be bound by an ADCC-inducing antibody (e.g., 3BNC117). We are utilizing the plasmid Q842.D16 to transfect HEK293T cells causing the cells to express HIV-1 envelope (gp160). We are using a non-ADCC antibody that does not compete with the ADCC antibody for a binding site (e.g., PG9) to identify HIV-Env positive target cells to measure their death in a killing assay. To make PG9 useful in this context, we labeled the antibody with a fluorophore that can be detected in the flow cytometer used to measure cell killing in our assay. The HIV Reagent Program graciously provided Q842.D16, PG9, and 3BNC117. Results Using a Nanodrop 2000, unlabeled PG9 was determined to have a protein concentration of 3.1E-4 mM and a degree of labeling (DOL) of 8.36 units. The DOL of PG9 was in the acceptable range (4-9 units). Initial target cell labeling experiments were encouraging. However, subsequent data suggest that PG9 is non-specifically binding to on the surface of 293T cells. Next steps are to find an appropriate blocking strategy for this step in the procedure such that we have reproducible target cell recognition in the killing assay. Discussion Despite the discouraging setback in target cells recognition, we are optimistic that this technical challenge will be overcome. For example, we are investigating an alternative strategy of identifying HIV-Env positive cells by having the envelope expression vector also express green fluorescent protein to bypass the need for a second antibody altogether. Details about this concept will also be presented in the poster. The work in this poster builds on the initial steps of the process described in the partner poster presented by Jaden Nienhueser

Sex in a material world: why the study of sexual reproduction and sex-specific traits should become more nutritionally-explicit

International audienceRecent advances in nutritional ecology, particularly arising from Ecological stoichiometry and the Geometric framework for nutrition, have resulted in greater theoretical coherence and increasingly incisive empirical methodologies that in combination allow for the consideration of nutrient-related processes at many levels of biological complexity. However, these advances have not been consistently integrated into the study of sexual differences in reproductive investment, despite contemporary emphasis on the material costs associated with sexually selected traits (e.g. condition-dependence of exaggerated ornaments). Nutritional ecology suggests that material costs related to sex-specific reproductive traits should be linked to quantifiable underlying differences in the relationship between individuals of each sex and their foods. Here, we argue that applying nutritionally-explicit thought to the study of sexual reproduction should both deepen current understanding of sex-specific phenomena and broaden the tractable frontiers of sexual selection research. In support of this general argument, we examine the causes and consequences of sex-specific nutritional differences, from food selection and nutrient processing to sex-specific reproductive traits. At each level of biological organization, we highlight how a nutritionally-explicit perspective may provide new insights and help to identify new directions. Based on predictions derived at the individual level, we then consider how sex-specific nutrient limitation might influence population growth, and thus potentially broader patterns of life history evolution, using a simple population dynamics model. We conclude by highlighting new avenues of research that may be more accessible from this integrative perspective

Development and implementation of natural killer cell simultaneous ADCC and direct killing assay

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing