Engineering novel functions in mammalian cells is crucial for biomedical research, revolutionizing cell-based diagnostics and therapeutics. Synthetic receptors have enabled the creation of designer cells capable of detecting and correcting disease states. However, improving the specificity of these approaches requires multilayered circuits for combinatorial biomarker detection. Large genetic circuits are challenging to implement in monocultures due to resource sharing limitations and the limited packaging capacity of vectors for multiple genetic constructs. To fully utilize this technology, engineered cells need reciprocal communication and information processing resembling specialized cell consortia like the human immune system. The human immune system is an ideal model for a mammalian synthetic communication network. It efficiently senses and responds to numerous diffusing signals, performs distributed computing, and leverages specialized cell types. Such a network can reduce resource competition by enabling distributed information processing, leading to advanced functions like population control and coordinated therapeutic cell responses. This contrasts with previous synthetic prokaryotic communication systems. Ideally, cells in such a network should sense orthogonal, soluble stimuli and produce user-defined responses. Previous work achieved synthetic, intercellular, juxtacrine communication using synthetic receptors performing logic operations based on cell-to-cell contact. Diffusion-based intercellular communication has been attempted using repurposed small molecules or directed evolution of natural proteins. However, directed evolution is labor-intensive, lacks inherent Boolean logic capabilities, and existing small molecule approaches lack scalability and receptor-level Boolean logic. While advancements have been made in synthetic communication between mammalian cells, a scalable, orthogonal, and receptor-level logic-capable platform using diffusible ligands has been missing. This work engineers such a platform.

The introduction comprehensively reviews existing methods for engineering synthetic communication in mammalian cells. It highlights the successes and limitations of prior approaches, including those using synthetic receptors, repurposed small molecules, and directed evolution of natural proteins. The review establishes the need for a scalable, orthogonal platform capable of performing Boolean logic operations at the receptor level, a gap that this research aims to fill. The discussion of the human immune system as a model highlights the complexity of natural cell-cell communication and underscores the ambition of the proposed synthetic system.

The researchers engineered a scalable and orthogonal synthetic communication platform for mammalian cells based on designed diffusible ligands. This platform utilizes the Generalized Extracellular Molecule Sensor (GEMS) platform, which allows for customized input and output. GEMS receptors consist of an erythropoietin receptor (EpoR) transmembrane scaffold fused to intracellular signaling domains. The extracellular EpoR domain was functionalized with coiled-coil peptides (CCs) that orthogonally bind to each other. Cognate CC pairing induces receptor heterodimerization and downstream signaling via the JAK/STAT, MAPK, PI3K/Akt, or PLCG pathways, leading to transgene expression. The study monitored CC-induced receptor activation using HEK293T cells transiently transfected with CC-GEMS receptors, STAT3, and a secreted alkaline phosphatase (SEAP) reporter gene. SEAP secretion, quantified using a colorimetric assay, indicated receptor activation. The researchers investigated the impact of linker length between the CC and EpoR domains on receptor activation. They also designed and synthesized soluble, ditopic CC peptide ligands (N-termini linked A'-A' dipeptides) to activate CC-GEMS receptors. These ligands were expressed in *E. coli*, purified, and tested for their ability to activate receptors. To enable intercellular communication, the researchers designed and expressed secreted SUMO-tagged CC dipeptides. They created sender cells secreting the ligands and receiver cells expressing cognate receptors and reporters. They demonstrated Boolean logic (AND and OR gates) using combinations of receptors and ligands. They also rerouted CC-GEMS signaling through the PLCG pathway and tested the system's ability to control the secretion of therapeutic proteins (IL-10). Three-cell population systems were constructed to perform distributed AND gate operations, and the researchers analyzed results via various techniques, including SDS-PAGE, Native-PAGE, SEC, confocal microscopy, flow cytometry, ELISA, and western blotting.

The study's key findings demonstrate the successful creation and validation of a novel synthetic communication platform (CC-GEMS) in mammalian cells. This platform utilizes coiled-coil peptides (CCs) as orthogonal signaling molecules. The findings include: 1. **Orthogonal Receptor Activation:** Cognate CC pairs robustly activate CC-GEMS receptors, while non-cognate pairs show negligible activation. This orthogonality is maintained across different linker lengths between the CC and EpoR domains, showcasing the robustness of the system. 2. **Soluble Ditopic CC Ligands:** Synthetic, soluble, ditopic CC ligands effectively activate cognate CC-GEMS receptors, demonstrating a method for triggering receptor activation through diffusible signals. The response showed a bell-shaped dose-response curve, suggesting tunability through ligand concentration. 3. **Secreted Ditopic CC Ligands:** The researchers successfully engineered a system where cells express and secrete ditopic CC ligands, enabling intercellular communication. This secretion can be controlled by a doxycycline-inducible promoter, providing an ON/OFF switch for communication. 4. **Boolean Logic Operations:** The CC-GEMS platform demonstrates both OR and AND gate logic operations at the receptor level. This capability is achieved through the use of different combinations of receptors and ligands and highlights the system's potential for complex computation. 5. **Intercellular Communication:** A three-cell population system, consisting of two sender populations secreting distinct ligands and a receiver population expressing a cognate receptor, successfully performed distributed AND gate operations, proving the system's applicability for complex information processing between multiple cell populations. 6. **Therapeutic Protein Expression:** The CC-GEMS platform successfully controlled the secretion of a therapeutic protein (IL-10) upon activation by a cognate CC dipeptide. This finding demonstrates the system's potential for therapeutic applications.

The CC-GEMS platform addresses the limitations of existing approaches to synthetic mammalian cell communication. Its scalability, orthogonality, and ability to perform Boolean logic operations at the receptor level represent significant advancements. The platform's modular design allows for customization and expansion, enabling the creation of complex cell consortia with diverse functionalities. The use of diffusible ligands avoids the limitations of contact-dependent systems and allows for distributed information processing. The successful implementation of Boolean logic gates at the receptor level enhances the system's computational capabilities. The results support the potential of CC-GEMS for various applications, including cell-based therapeutics and diagnostics. Future research should explore the optimization of receptor density for improved signaling, expanding the platform's compatibility with other receptor architectures, and in vivo testing in relevant animal models. The platform could be applied to develop advanced cell therapies and diagnostic tools, improving their specificity and efficiency.

This research presents CC-GEMS, a novel and versatile platform for engineering synthetic communication in mammalian cells. The platform successfully demonstrates orthogonal receptor activation, soluble and secreted ligand-mediated activation, Boolean logic operations (OR and AND gates), intercellular communication, and therapeutic protein expression. Its modular design and scalability make it a powerful tool for creating sophisticated cell-based systems with diverse applications in therapeutics and diagnostics. Future work should focus on further optimizing the platform, exploring its use in primary immune cells, and conducting in vivo studies.

While the CC-GEMS platform shows considerable promise, there are some limitations to consider. The study primarily used HEK293T cells, which may not fully reflect the behavior of other cell types. Although the system demonstrated robust control of protein secretion, the level of activation in mammalian-expressed secreted ligands was lower compared to those expressed in bacteria. Further optimization of the system for different cell types and exploring the use of different promoters and reporter systems could enhance its versatility and applicability to a wider range of research questions and therapeutic contexts.