The DNA damage response (DDR) encompasses a multitude of interconnected pathways that serve as a cellular defense to protect genome integrity. Dysregulation or failure of these pathways results in cancers and genetic disease. DNA viruses, including the herpesvirus cytomegalovirus (CMV), activate DDR signaling during their replicative program. The mechanisms by which they commandeer these responses for replication of their genome remain unclear. Here, we define a viral protein, UL138, that modulates the activity of host DDR pathways. The loss of UL138 results in structural variants, including inversions, deletions, and duplications, with signature of homology-directed repair and other DDR pathways. The actions of UL138 are due, in part, to its modulation of pathways regulated by the cellular deubiquitinating complex that targets proliferating cell nuclear antigen (PCNA) and Fanconi Anemia effectors, FANCD2 and FANCI. However, we also show that UL138 accesses pathways independent of USP1-PCNA/FANCD2/FANCI. Disruption of UL138 or these pathways impacted viral genome replication and had consequences for viral genome integrity. This work provides mechanistic insight into the long-standing questions of how DNA viruses recruit, modulate and use cellular DDR pathways. It also puts forth CMV as a model system for further defining these pathways in human cells.

The human cell cycle is a highly regulated process that integrates multiple signaling pathways and checkpoints to ensure faithful genome duplication and cell division. Disruptions in these regulatory networks contribute to a wide range of diseases. Here, we present a novel, updateable computational model of the full human cell cycle that shows sustained oscillations over time and reproduces experimental perturbations. We used a hybrid framework combining mass action and Michaelis–Menten kinetics, incorporating the synthesis, degradation, and regulation of key cell cycle proteins and protein complexes. It consists of 63 distinct biochemical species, interacting through 41 major reactions, and functioning through 63 ODEs. The model is built upon a modular framework, structured around the core regulatory networks of the G1, S, G2, and M phases. Due to its complexity, we determined parameter sets that met strict criteria, namely event timing, comparable concentrations, and continuous cycling. We validated the model’s behavior by reproducing canonical check-point responses, including mitogen dependence and the DNA damage response, both of which produced reversible and robust cell cycle arrests. Importantly, the model was trained and calibrated using in vitro data from human U251-MG glioma cells expressing the FastFUCCI cell cycle re-porter. We quantitatively aligned the simulated and experimentally determined phase durations and cell doubling times. Next, we experimentally tested and refined model parameters by using abemaciclib-mediated inhibition of CDK4 and volasertib-mediated inhibition of PLK1. In vitro and in silico data show dose-dependent G1 arrest by abemaciclib and dose-dependent mitotic arrest by volasertib. Finally, we demonstrated that the model predicts changes in cell proliferation over a wide range of drug concentrations and combinations. Overall, our work establishes a robust, data-driven computational model for systems-level analysis of the human cell cycle and its disruption by therapeutic perturbations. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionThe complex dynamics of chimeric antigen receptor T-cell (CAR-T cell) cytotoxicity and proliferation are potential factors that influence the clinical response to CAR-T therapy. The patient-specific functionality of CAR-T products play a role in these dynamics. CAR-T products comprise phenotypically and functionally distinct populations of cells that impact therapy response in different ways. We hypothesized that product-specific parameters exist that predict individual patient responses to therapy and that these can be elucidated by simulating the interactions of CAR-T products and tumor cells using an in vitro assay-based model.MethodsWe use an ordinary differential equation (ODE)-based pharmacokinetic (PK) and pharmacodynamic (PD) model to characterize key CAR-T cell functional parameters. Parameters for the model developed using our method are product-specific and derived from in vitro assays performed on individual patient CAR-T products from clinical trial NCT04186520.ResultsOur results demonstrate that while considerable variability is present in in vitro cytotoxicity kinetics and subsequently estimated model parameters between each product, these differences do not predict early (28 days) or late responses (90 days) after treatment across the total cohort of patients investigated. However, we show that differences in an estimated model parameter for increased CAR-T cell responsiveness to tumor cytotoxicity are correlated with durable therapy responses (no relapse through 180 days). Additionally, in a cohort of diffuse large B-cell lymphoma (DLBCL) patients, we demonstrate that a model parameter estimating cooperativity between CAR-T cells is also correlated with durable therapy responses and that may be related to differences in CD4:CD8 ratios in the CAR-T cell product.ConclusionsOverall, our work demonstrates that while pre-treatment CAR-T cell functional parameters vary on a patient and product basis, these parameters do not predict initial therapeutic responses. We find that initial therapeutic responses are possible across a range of initial product kinetic parameters. However, we observed that their potentially exist unique kinetic properties associated with the initial product that is predictive of disease relapse.

Objectives/Goals: Congenital cytomegalovirus (cCMV) continues to be the primary infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells in response to cCMV remains largely unexplored. This study seeks to investigate how fetal NK cells respond to human cytomegalovirus (HCMV) during gestation. Methods/Study Population: Umbilical cord blood and corresponding umbilical cord tissues were collected from fetuses that had no complications during gestation. These samples, provided by the Medical College of Wisconsin Tissue Bank, were processed within 24 hours after live birth. Single-cell suspensions were prepared from the samples, and fetal NK cells were isolated and exposed to HCMV antigen peptides VMAPRTLFL, VMAPRTLIL, VMAPQSLLL, and the human self-peptide ALALVRMLI. These peptides were presented on HLA-E*01:03 BV421-conjugated tetramers produced by the National Institutes of Health Tetramer Core Facility. Additionally, fetal NK cells were also prepared for single-cell RNA sequencing (scRNA-seq), and cells were filtered and clustered based on the number of uniquely expressed genes. Results/Anticipated Results: Through unbiased clustering, our scRNA-seq analysis identified five unique fetal NK cell subsets in umbilical cord blood and four in the corresponding umbilical cord tissue. Notably, fetal NK cells exposed to HCMV during gestation were primarily mature NK cell subsets, while those from unexposed fetuses were mostly immature subsets. Additionally, HCMV-exposed fetal NK cells exhibited a strong recall response to the HCMV antigen, with a notably higher frequency and elevated production of IFN-γ. Conversely, naïve fetal NK cells from fetuses unexposed to HCMV produced significantly lower levels of IFN-γ. Finally, we identified a distinct subset of fetal NK cells that emerge following exposure to the HCMV antigen. Discussion/Significance of Impact: In this study, we show that HCMV infection can influence the formation of specific NK cell subsets and re-exposure to the HCMV antigen can trigger a recall response. These insights could pave the way for the development of innovative NK cell-based immunotherapies aimed at preventing fetuses from developing symptomatic cCMV.

Human cytomegalovirus (HCMV) is a prolific human herpesvirus that infects most individuals by adulthood. While typically asymptomatic in adults, congenital infection can induce serious neurological symptoms, including hearing loss, visual deficits, cognitive impairment, and microcephaly in 10%-15% of cases. HCMV has been shown to infect most neural cells, with our group recently demonstrating this capacity in stem cell-derived forebrain neurons. Infection of neurons induces deleterious effects on calcium dynamics and electrophysiological function paired with gross restructuring of neuronal morphology. Here, we utilize an induced pluripotent stem cell-derived model of the human forebrain to demonstrate how HCMV infection induces syncytia, drives neurite retraction, and remodels microtubule networks to promote viral production and release. We establish that HCMV downregulates microtubule-associated proteins while largely sparing other cytoskeletal elements. Furthermore, we pharmacologically modulate microtubule dynamics using paclitaxel (stabilize) and colchicine (destabilize) to examine the effects on neurite structure, syncytial morphology, and viral release. With paclitaxel, we found improvement of neurite outgrowth, but neither paclitaxel nor colchicine impacted viral titers. Together, these data suggest that HCMV infection-induced disruption of microtubules in human cortical neurons can be partially mitigated with microtubule stabilization, suggesting a potential avenue for future neuroprotective strategies.IMPORTANCEInfection by human cytomegalovirus (HCMV) continues to cause significant damage to human health. In the absence of a vaccine, vertical transmission from mother to fetus can result in profound neurological damage impacting quality of life. These studies focus on understanding the impact of HCMV infection on forebrain cortical neurons derived from induced pluripotent stem cells (iPSCs). We show that infection results in loss of neurite extension accompanied by cell-to-cell fusion. These pathogenic changes involve HCMV infection-mediated disruption of the microtubule network in iPSCs from different patient backgrounds. The microtubule stabilization agent paclitaxel partially protected neurite length and altered syncytia morphology without impacting viral replication. This work is part of our continued efforts to define putative strategies to limit HCMV-induced neurological damage.

Summary:Molecular mechanisms of biological functions and disease processes are exceptionally complex, and our ability to interrogate and understand relationships is becoming increasingly dependent on the use of computational modeling. We have developed "BioModME," a standalone R-based web application package, providing an intuitive and comprehensive graphical user interface to help investigators build, solve, visualize, and analyze computational models of complex biological systems. Some important features of the application package include multi-region system modeling, custom reaction rate laws and equations, unit conversion, model parameter estimation utilizing experimental data, and import and export of model information in the Systems Biology Matkup Language format. The users can also export models to MATLAB, R, and Python languages and the equations to LaTeX and Mathematical Markup Language formats. Other important features include an online model development platform, multi-modality visualization tool, and efficient numerical solvers for differential-algebraic equations and optimization.Availability and implementation:All relevant software information including documentation and tutorials can be found at https://mcw.marquette.edu/biomedical-engineering/computational-systems-biology-lab/biomodme.php. Deployed software can be accessed at https://biomodme.ctsi.mcw.edu/. Source code is freely available for download at https://github.com/MCWComputationalBiologyLab/BioModME.

ABSTRACT Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual’s life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities, which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing multiplicity of infection (MOI) in silico , we observed near-identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection. IMPORTANCE Human cytomegalovirus (HCMV) infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live-cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection, which we define as the effective multiplicity of infection, and this subset would otherwise be missed when analyzing the average of the population.

ABSTRACTHuman cytomegalovirus (HCMV) is a prevalent betaherpesvirus, and infection can lead to a range of symptomatology from mononucleosis to sepsis in immunocompromised individuals. HCMV is also the leading viral cause of congenital birth defects. Lytic replication is supported by many cell types with different kinetics and efficiencies leading to a plethora of pathologies. The goal of these studies was to elucidate HCMV replication efficiencies for viruses produced on different cell types upon infection of epithelial cells by combining experimental approaches with data-driven computational modeling. HCMV was generated from a common genetic background of TB40-BAC4, propagated on fibroblasts (TB40Fb) or epithelial cells (TB40Epi), and used to infect epithelial cells. We quantified cell-associated viral genomes (vDNA), protein levels (pUL44, pp28), and cell-free titers over time for each virus at different multiplicities of infection. We combined experimental quantification with data-driven simulations and determined that parameters describing vDNA synthesis were similar between sources. We found that pUL44 accumulation was higher in TB40Fbthan TB40Epi. In contrast, pp28 accumulation was higher in TB40Epiwhich coincided with a significant increase in titer for TB40Epiover TB40Fb. These differences were most evident during live-cell imaging, which revealed syncytia-like formation during infection by TB40Epi. Simulations of the late lytic replication cycle yielded a larger synthesis constant for pp28 in TB40Epialong with increase in virus output despite similar rates of genome synthesis. By combining experimental and computational modeling approaches, our studies demonstrate that the cellular source of propagated virus impacts viral replication efficiency in target cell types.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause serious disease under conditions of immunodeficiency and upon congenital infection. HCMV replicates in diverse cell types throughout the human body with tropism influenced by the source of the virus. Here, we investigated the contribution of viral sources to the kinetics of HCMV replication in epithelial cells using both experimental and mechanistic computational modeling approaches. These studies reveal that HCMV produced from epithelial cells exhibits a higher efficiency of replication despite similar viral DNA synthesis kinetics between viral sources. These differences likely involve a propensity of epithelial-derived virus to induce syncytia versus fibroblast-derived virus, and an accompanying higher synthesis rate of a late virion protein ultimately resulting in production of more extracellular infectious virus.

Human cytomegalovirus (HCMV) is a prolific human herpesvirus that infects most individuals by adulthood. While typically asymptomatic in adults, congenital infection can induce serious neurological symptoms including hearing loss, visual deficits, cognitive impairment, and microcephaly in 10-15% of cases. HCMV has been shown to infect most neural cells with our group recently demonstrating this capacity in stem cell-derived forebrain neurons. Infection of neurons induces deleterious effects on calcium dynamics and electrophysiological function paired with gross restructuring of neuronal morphology. Here, we utilize an iPSC-derived model of the human forebrain to demonstrate how HCMV infection induces syncytia, drives neurite retraction, and remodels microtubule networks to promote viral production and release. We establish that HCMV downregulates microtubule associated proteins at 14 days postinfection while simultaneously sparing other cytoskeletal elements, and this includes HCMV-driven alterations to microtubule stability. Further, we pharmacologically modulate microtubule dynamics using paclitaxel (stabilize) and colchicine (destabilize) to examine the effects on neurite structure, syncytial morphology, assembly compartment formation, and viral release. With paclitaxel, we found improvement of neurite outgrowth with a corresponding disruption to HCMV-induced syncytia formation and Golgi network disruptions but with limited impact on viral titers. Together, these data suggest that HCMV infection-induced disruption of microtubules in human cortical neurons can be partially mitigated with microtubule stabilization, suggesting a potential avenue for future neuroprotective therapeutic exploration.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multidrug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; V-max) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a six-parameter version of this model to control time-course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only three unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT, found the mean estimated value for each parameter, and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of V-max for MRP2 at the 30-min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP. NEW & NOTEWORTHY We developed a computational model of sodium fluorescein (SF) biliary excretion in ex vivo machine perfusion and used this model to assess changes in model parameters associated with the activity of MRP2, a hepatocyte membrane transporter, in response to increasing warm ischemia time. We found a significant decrease in the parameter value describing MRP2 activity, consistent with a role of decreased MRP2 function in ischemia-reperfusion injury leading to decreased secretion of SF into bile.

Background: Natural Killer (NK) cells play a crucial role in the immediate immune response against virally infected and transformed cells. Managing human cytomegalovirus (HCMV) infections relies on a subset of NKG2C+ NK cells. The NKG2C/CD94 receptor complex identifies and responds to HCMV-infected cells expressing HLA-E loaded with viral gpUL40 antigen peptide. When activated, NKG2C+ NK cells exhibit a response similar to adaptive immunity and establish a reservoir of memory cells. As a result, HCMV+ individuals have significantly elevated levels of long-lived Memory NKG2C+ NK cells that persist within the host. The long-term residency of memory NK cells and their developmental and functional outcomes following re-exposure to the HCMV gpUL40 antigen remains largely unknown. In our current study, we identify a unique subset of Memory NKG2C+ NK cells within the human spleen that expand and increase their effector function capabilities upon re-exposure to the HCMV gpUL40 antigen peptide. Methods & Results: Human spleens were obtained from healthy adult donors, both HCMV+ and HCMV-. These spleens were provided by the Versiti Organ Donor Center of Wisconsin and tissues were processed into single-cell suspensions. NKG2C+ NK cells were isolated and then incubated with viral antigen peptides VMAPRTLFL, VMAPRTLIL, VMAPQSLLL and the human self-peptide ALALVRMLI. The peptides VMAPRTLFL, VMAPRTLIL, and VMAPQSLLL haven been previously identified as clinical isolates of the HCMV gpUL4015-23 peptide. The human self-peptide ALALVRMLI derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 (MRP7), was used as a control, as previous reports have demonstrated it inhibits NK cell effector functions. These peptides were loaded on HLA-E*01:03 BV421-conjugated tetramers produced by the National Institutes of Health Tetramer Core Facility. We observed that tetramer staining of splenic NKG2C+ NK cells revealed positive binding of all four HLA-E tetramer/peptide complexes, with HLA-EVMAPRTLFL showing the highest proportion of tetramer-positive NKG2C+ cells. To verify specificity to the NKG2C receptor, we performed NKG2C receptor blockage prior to HLA-E tetramer staining and found a significant reduction in tetramer-positive NKG2C+ NK cells. We suspected the remaining tetramer-positive cells were NKG2C+ NKG2A+ NK cells, as this subset constitutes a small percentage of the total NKG2C+ NK cell population. Our findings confirmed that nearly all gated NKG2C+NKG2A+ NK cells were positive for the individual HLA-E tetramer/peptide complexes, suggesting that the NKG2A receptor binds with lower affinity. Notably, the HLA-EALALVRMLI tetramer/peptide complex, known to engage the NKG2A receptor with higher affinity, had the highest proportion of tetramer-positive cells. Collectively, these findings indicate our designed HLA-E tetramer/peptide complexes can specifically bind to NKG2C+ NK cells with varying affinities, and that a small subset of NKG2C+NKG2A+ NK cells can also bind to these HLA-E tetramer/peptide complexes with lower affinity via the NKG2A/CD94 receptor complex. To investigate if Memory NKG2C+ NK cells undergo antigen-specific expansion, we performed HLA-E tetramer incubation assays using individual HLA-E tetramer/peptide complexes. Importantly, HLA-EVMAPRTLFL resulted in the most significant expansion in the total proportion of NKG2C+ NK cells from both the HCMV+ and HCMV- donors, with a greater increase in the HCMV+ donors. In addition, Memory NKG2C+ NK cells displayed a strong recall response to the HCMV gpUL40 antigen, resulting in significantly higher total frequency and production of IFN-g. In contrast, naïve NKG2C+ NK cells from HCMV- donors developed a memory-like phenotype following incubation with the HCMV gpUL40 antigen. Together, our studies reveal Memory NKG2C+ NK cells isolated from the human spleen of HCMV+ donors mount a strong recall response to the HCMV gpUL40 antigen, and that naïve NKG2C+ NK cells can develop a memory-like phenotype and functional response following exposure to the HCMV gpUL40 antigen. Conclusion: Here, we find a distinct subset of long-lived Memory NKG2C+ NK cells that persist within the human spleen and demonstrate that the HCMV gpUL40 antigen can induce the formation and expansion of these cells. These findings can contribute to the future isolation and design of Memory NKG2C+ NK cell immunotherapies.

Since the FDA’s approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.

OBJECTIVES/GOALS: Congenital cytomegalovirus (cCMV) remains to be the leading infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells during cCMV remains largely unknown. The objective of this study is to define the transcriptomes of fetal NK cells exposed to human cytomegalovirus (HCMV) infection during gestation. METHODS/STUDY POPULATION: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV seropositive (HCMV+) and two HCMV seronegative (HCMV-) fetuses that did not experience any complications during gestation. These samples were provided by the Medical College of Wisconsin Tissue Bank and were processed within 24 hours following live birth. CD7+ CD3e-CD14-CD19-CD20- fetal NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. RESULTS/ANTICIPATED RESULTS: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV+ and two HCMV- fetuses. We were able to successfully sort and capture fetal NK cells and perform scRNA-seq on these samples. Following unbiased clustering, we observed and characterized five distinct fetal NK cell subsets in the umbilical cord blood and four fetal NK cell subsets in the corresponding umbilical cord tissue. Our findings revealed that HCMV+ fetal NK cells primarily consisted of mature NK cell subsets, while HCMV- fetal NK cells constituted the majority of the immature subsets. Importantly, we identified a unique subset of NKG2CHi fetal NK cells that were significantly elevated in the HCMV+ fetuses. Finally, we defined a group of transcription factors involved in the formation of antiviral fetal NK. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of distinct NK cell subsets and drive their unique transcriptional profiles. These findings have the potential to guide the development of an innovative NK cell immunotherapy that could help prevent fetuses from developing symptomatic cCMV.

Abstract Congenital cytomegalovirus (cCMV) infection is the most common cause of intrauterine infection in the USA, impacting 1 in 100 live births. Although most infected newborns are asymptomatic, 10% display severe congenital anomalies, including microcephaly, sensorineural hearing loss, cerebral palsy, growth restriction, and perinatal mortality. Natural killer (NK) cells are the first lymphocytes to develop during gestation and are required to manage CMV infections. The role of fetal NK cells during cCMV is limited and their development and functions within the umbilical cord has not yet been explored. To investigate this, four sets of umbilical cord blood (UCB) and matching umbilical cord tissues were collected from 2 CMV seropositive (CMV+) and 2 CMV seronegative (CMV-) fetuses that did not experience any complications during gestation. We were able to successfully sort and capture over 5,000 fetal NK cells and perform single-cell RNA-sequencing (scRNA-seq). Using scRNA-seq, we observed and characterized 5 unique fetal NK cell subsets in the UCB and 4 subsets in the corresponding tissue. Interestingly, we identified a distinct subset of NKG2C+ memory fetal NK cells displaying elevated expression levels of NKG2C, CD52, CD2, CD16, and CD3E. Here, we demonstrate that cCMV can induce the formation of memory-like NKG2C+ NK cells that display a unique transcriptional profile. These findings have the potential to influence the future application of fetal memory NK cells.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

Introduction: Human cytomegalovirus (HCMV) is a b-herpesvirus that is highly prevalent in the adult population and has the ability to establish lifelong latency in healthy individuals. The innate and adaptive immune systems work closely to control viral replication, leading to a dynamic interaction between the virus and the host immune system. This interplay leads to the development of distinct immune-cell repertoires in HCMV seropositive (HCMV +) individuals. Natural killer (NK) cells are cytotoxic innate lymphocytes that are required to manage and control HCMV infections. NK cells utilize their NKG2C/CD94 receptor complex to mount a response to infected cells that present HLA-E molecules loaded with the HCMV UL40-derived peptide. As a result, HCMV + individuals possess higher levels of NKG2C + NK cells, and within this population, there is a distinct subset referred to as “memory” due to their adaptive-like characteristics. The developmental origins and molecular mechanisms involved in the maintenance and persistence of these cells remain largely unknown. In our current study, we investigate the origins and transcriptional signatures of persistent memory NKG2C + NK cells obtained from human donor spleens. Methods & Results: Eight healthy adult human spleens were obtained from four HCMV + and four HCMV seronegative (HCMV -) donors. Donor median age was 59 [IQR 48.5-56.5], 50% (n=4) were identified as female, 50% (n=2) of females were HCMV + and 50% (n=2) of females were HCMV -. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. In line with previous findings in the peripheral blood of HCMV + individuals, we observed significantly elevated levels of NKG2C + NK cells in the spleens of HCMV + donors ( Fig. 1A). This observed significance of higher NKG2C + NK cells was consistent across all HCMV + donors when compared to our HCMV - donors ( Fig. 1A). To investigate the molecular mechanisms involved in the maintenance and persistence of memory NKG2C + NK cells, we performed single-cell RNA sequencing (scRNA-seq), using the sorted NK cells from all eight donors. Using unbiased clustering analysis, we identified and characterized four distinct NKG2C + splenic NK cell subsets ( Fig. 1B). Our findings indicated that the relative composition of these subsets was highly influenced by the HCMV status of the donors. Specifically, we observed that HCMV + donors had significantly higher levels of NKG2C Hi memory NK cells ( Fig. 1B). This NKG2C Hi memory NK subset had significantly higher expression of NKG2C, CD52, CD3 e, and IL7R ( Fig. 1C) and significantly lower expression of FCER1G, KLRC1, CD247, ZBTB16, SYK, and SH2D1B ( Fig. 1C). These findings suggest NKG2C Hi memory NK cells possess unique transcriptional and molecular mechanisms that may contribute to their ability to persist over time. To explore the developmental cell fate of NKG2C Hi memory NK, we utilized both the Monocle 2 and Monocle 3 software's to track how NKG2C + cells transition between transcriptomic states. Monocle 3 analysis yielded a simple early-to-late cell fate trajectory that was projected onto our UMAP plot ( Fig. 2A). We found that NKG2A Hi NKG2C + subset served as the early timepoint in the developmental trajectory, ultimately leading to the development of NKG2C Hi memory NK ( Fig. 2A). The Monocle 2 analysis produced unique developmental trajectory plots, allowing us to visualize distinct developmental pathways and transitions ( Fig. 2B). Interestingly, we observed a unique branch-point exclusive to the HCMV + donors ( Fig. 2B) that expressed significantly higher levels of CD3 e and IL7R. These observations could indicate a specific developmental pathway unique to NKG2C Hi memory NK. Conclusion: Here, we demonstrate that HCMV infection can induce the formation of NKG2C Hi memory NK cell subset that displays a unique transcriptional and developmental profile. These findings can influence the future isolation and application of memory NK cells in cellular immunotherapies.

OBJECTIVES/GOALS: The primary objective of this study was to define the transcriptomes and transcriptional regulatory network required for the development and function of adaptive Natural Killer (NK) cells in donors with latent human cytomegalovirus (HCMV) infection. METHODS/STUDY POPULATION: Eight healthy adult human spleens were obtained from four HCMV seropositive and four HCMV seronegative donors. Spleens were provided by the Versiti Organ Donor Center of Wisconsin and were processed to a single cell suspension. CD7+ CD3E- CD14- CD19- CD20- NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. The monocle software was used for single cell trajectory analysis and the SCENIC software was used to decipher gene regulatory networks. RESULTS/ANTICIPATED RESULTS: Eight healthy spleens from four HCMV seropositive and four HCMV seronegative donors were obtained and their NK cells were sorted and captured for scRNA-seq. Donor median age was 59 [IQR 48.5-56.5], 50% (n=4) were female and all donors were not experiencing any acute or chronic symptoms. Using scRNA-seq, we observed elevated numbers of NKG2C+ adaptive NK cells in HCMV seropositive individuals when compared to HCMV seronegative individuals. In addition, we identify a set of transcription markers and regulators that are responsible for the development and function of adaptive NKG2C+ NK cells. Finally, our trajectory analysis of adaptive NKG2C+ NK cells revealed a unique developmental pathway. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C+ NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies.

Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that typically presents asymptomatically in healthy individuals despite lifelong latency. However, in 10-15% of congenital cases, this beta-herpesvirus demonstrates direct effects on the central nervous system, including microcephaly, cognitive/learning delays, and hearing deficits. HCMV has been widely shown to infect neural progenitor cells, but the permissiveness of fully differentiated neurons to HCMV is controversial and chronically understudied, despite potential associations between HCMV infection with neurodegenerative conditions. Using a model system representative of the human forebrain, we demonstrate that induced pluripotent stem cell (iPSC)-derived, excitatory glutamatergic and inhibitory GABAergic neurons are fully permissive to HCMV, demonstrating complete viral replication, competent virion production, and spread within the culture. Interestingly, while cell proliferation was not induced in these post-mitotic neurons, HCMV did increase expression of proliferative markers Ki67 and PCNA suggesting alterations in cell cycle machinery. These finding are consistent with previous HCMV-mediated changes in various cell types and implicate the virus' ability to alter proliferative pathways to promote virion production. HCMV also induces significant structural changes in forebrain neurons, such as the formation of syncytia and retraction of neurites. Finally, we demonstrate that HCMV disrupts calcium signaling and decreases neurotransmission, with action potential generation effectively silenced after 15 days post infection. Taken together, our data highlight the potential for forebrain neurons to be permissive to HCMV infection in the CNS, which could have significant implications on overall brain health and function.