Biology subjects

Beckett, A. N.

Publications and source records attributed to Beckett, A. N..

4 recordsLinked to original sources

bioRxiv2026

Programmable synthetic cytokine receptors polarize macrophages to user-defined functional states

Technology that precisely controls macrophage polarization to distinct functional states would deepen our understanding of macrophage biology and enable the development of new macrophage cell therapies. Here, we use a synthetic cytokine receptor (SCR) platform with a programmable signaling domain to control the polarization of primary human macrophages. SCRs containing signaling motifs from the interferon-gamma (IFN-{gamma}) or Interleukin-10 (IL-10) receptors mimic key features of pro-inflammatory or anti-inflammatory polarization, respectively. Random recombination of nine distinct signaling motifs to create new SCR signaling domains generates a diverse landscape of synthetic macrophage states with varied expression of inflammatory markers (CD80, CD40) and anti-inflammatory markers (CD163, CD206), and varied phagocytic capacity. SCRs programmed with multiple YLxQ motifs increase macrophage phagocytosis of E. coli and chimeric antigen receptor (CAR)-macrophage phagocytosis of cancer cells in mice, reducing tumor burden by 30-fold. The motif-dependent polarization is well-described by a two-state model, enabling quantitative prediction of macrophage polarization state from SCR signaling domain composition. Leveraging this model, we design an SCR that simultaneously enhances phagocytosis and maintains a macrophage pro-inflammatory state. Together, these findings establish a framework for synthetic programming of macrophage polarization states, with potential applications in cancer immunotherapy and other disease contexts.

synthetic biology↗

bioRxiv2025

A continuous landscape of signaling encodes a corresponding landscape of CAR T cell phenotype

Cytokine signaling is critical to the function of natural immune cells and engineered immune cell therapies such as chimeric antigen receptor (CAR) T cells. It remains unclear how the limited set of signal transducers and activators of transcription (STATs) and other proteins activated by these cytokine receptors can encode the observed diversity of immune cell phenotypes. To understand how signaling downstream of cytokines control immune cell phenotype, we sought to map the structure of Janus kinase (JAK)/STAT signaling domains to cell signaling and resulting CAR T cell function. We recombined 14 signaling motifs to construct a library of [~]30,000 constitutively active synthetic cytokine receptors (SCRs) with intracellular domains composed of novel signaling motif combinations that activate different signaling cascades. We experimentally tested [~]450 SCRs which generated a range of CAR T cell memory, cytotoxicity, and proliferation. SCRs with pSTAT1 and 3 signaling generated effector memory CAR T cells, while SCRs with strong pSTAT5 generated effector CAR T cells with potent anti-tumor activity. To map the structure-signaling-phenotype landscape we trained models to predict signaling and CAR T cell phenotype that result from varied motif combinations. From neural network predictions we identified features, including strong STAT5 and Shc signaling, that promote unsafe autonomous CAR T cell proliferation. Models also revealed a trade-off between memory and cytotoxicity, with a Pareto front encoded by a continuous change in signaling. These results demonstrate that recombination of a limited set of signaling motifs creates a continuous spectrum of signaling that encodes a corresponding spectrum of cell phenotype. This work synthetically expands the combinatorial space of JAK/STAT signaling and provides a foundation for rational design of CAR T cells with improved cytotoxicity, memory, and safety profiles. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/658149v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@cc1498org.highwire.dtl.DTLVardef@5c2864org.highwire.dtl.DTLVardef@5fc8b3org.highwire.dtl.DTLVardef@719f8f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Structural analyses of apolipoprotein A-IV polymorphisms Q360H and T347S elucidate the inhibitory effect against thrombosis

Apolipoprotein A-IV (apoA-IV) is an abundant lipid-binding protein in blood plasma. We previously reported that apoA-IV, as an endogenous inhibitor, competitively binds platelet IIb{beta}3 integrin from its N-terminal residues, reducing the potential risk of thrombosis. This study aims to investigate how the apoA-IVQ360H and apoA-IVT347S mutations affect the structure and function of apoA-IV. These mutations are linked to increased risk of cardiovascular diseases due to multiple single-nucleotide polymorphisms in the C-terminal region of apoA-IV. We postulate the structural hindrance caused by the C-terminal motifs may impede the binding of apoA-IV to platelets at its N-terminal binding site. However, the mechanistic impact of Q360H and T347S polymorphisms on this intermolecular interaction and their potential contribution to the development of cardiovascular disease have not been adequately investigated. To address this, recombinant forms of human apoA-IVWT, apoA-IVQ360H, apoA-IVT347S variants were produced, and the structural stability, dimerization, and molecular dynamics of the C-terminus were examined utilizing biophysical techniques including fluorescence anisotropy, fluorescence spectrophotometry, circular dichroism, and biolayer interferometry methods. Our results showed a deceased fraction of -helix structure in apoA-IVQ360H and apoA-IVT347S compared to the wildtype, and the inhibitory effect of dimerized apoA-IV on platelet aggregation was reduced in apoA- IVQ360H and apoA-IVT347S variants. Binding kinetics of examined apoA-IV polymorphisms to platelet IIb{beta}3 suggest a potential mechanism for increased risk of cardiovascular diseases in individuals with apoA-IVQ360H and apoA-IVT347S polymorphisms.

biochemistry↗

bioRxiv2023

A high-content screen identified ingenol-3-angelate as an enhancer of B7-H3-CAR T cell activity by increasing B7-H3 protein expression on the target cell surface via protein kinase C alpha activation

CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches may ultimately be required to achieve widespread clinical success. In this regard, we developed a novel high-content and high-throughput screen to evaluate 1114 FDA approved drugs to increase expression of the solid tumor antigen B7-H3 in metastatic osteosarcoma cells. In this proof-of-principle screen, we demonstrate that ingenol-3-angelate increased B7-H3 (CD276) mRNA, total protein, and cell surface expression. Mechanistically, ingenol-3-angelate increased B7-H3 expression via protein kinase C alpha activation. Functionally, ingenol-3-angelate induced B7-H3 expression enhanced B7-H3-CAR T cell function, highlighting utility of the approach, and paving the way for expanding this high-throughput and high-content technique to study other tumor and CAR T cell combinations.

cancer biology↗