(J-378) Redirecting Type I Interferon Signal Flow Through Mesoscale Clustering and De Novo Activation

Inflammatory cytokines such as type I interferons (type I IFN) play a controversial role in cancer biology. At the early stage, activation of type I IFNs is critical for anti-tumor immune responses; however, constitutive activation of type I IFN leads to T cell dysfunction and is known to contribute to cancer cell resistance to immune checkpoint blockade (ICB) treatment. Currently, strategies such as de novo-designed signaling molecules or small-molecule drugs are used to regulate type I IFN signaling at a limited efficiency with the risks of off-target effects and cytokine release syndrome. Here, we propose to use intrinsically disordered proteins (IDPs) to construct protein circuits that inhibit and redirect type I IFN activity through clustering and de novo activation of relevant Janus Kinases and signal transducer and activator of transcription proteins (JAK-STAT). Upon completion of the proposed goal, our approach will allow dynamic temporal regulation and rewiring of the excess type I IFN signaling to type II IFN signaling which is critical for T cell functions with high specificity and improved efficiency. It can be employed independently or synergistically with existing methods to improve the current immune therapies.  

Method: Genes encoded synthetic scaffolds were cloned into either pcDNA3.1 vector (Addgene) for transient expression or 3^rd gen lentiviral vector pLJM1 (Addgene) for lentiviral transduction. The inducible versions of the scaffold were generated by swapping the constitutive promoter to either Tet-on 3G promoter (Takara Bio). Transient transfections were performed through lipofection via lipofectamine 2000 (Invitrogen). Lentivirus were produced by lipofection in HEK293T cells. All cell lines (HEK293T, U2OS, Jurkat T cells) used in this study were obtained from ATCC. Primary T cells (CD4+/CD8+) were isolated from patients’ PBMCs and obtained through the Penn immunology core. HEK293T and U2OS Cells were maintained in EMEM with plasmocin and FBS. Jurkat T cells and primary T cells are cultured in RPMI1640 with IL-2.  Cell lines with CRISPR-tagged dimerization motifs at the C-terminus of endogenous target kinases were FACS single-sorted into 96 well plates and expanded from a single cell. Evaluation of condensate formation, recruitment, enrichment, and client partitioning were primarily done by data acquisition from confocal microscopy followed by analysis using ImageJ as described previously¹. For evaluating the efficiency of inducible systems, induced and uninduced cells were run through flow cytometry, followed by data analysis on FlowJo. Optogenetic release of the client were done by 10 sec 405nm light pulse in a 40 sec acquisition cycle for consecutively 11 cycles.

We observed that up to 80% of scaffold proteins and up to 60% of endogenous clients ended up in the synthetic organelles via cognate recruitment motifs and no recruitment with non-matching recruitment motifs, demonstrating efficient and specific client recruitment. Lentiviral generated cells showed slightly weaker scaffold and client partitioning with greater consistency compared to transient transfection. The optogenetic-controlled scaffold shows more than 50% of client release within the first 10 minutes, showing robust reversibility of the system controlled by light. Erk1 and Par6 double knock-in cell lines were screened and selected from single colony expansion and validated by PCR. We observed similar scaffold and client partitioning in the double knock-in cell lines with indications of phenotypical changes. We observed scaffold expression and JAKs recruitment in Jurkat T cells and primary T cells with modest efficiency. Our synthetic organelles have shown highly specific and efficient recruitment of endogenous clients with optogenetic controlled reversibility in standard cell lines, revealing its potential as a generic tool kit for cellular engineering. The future direction for this project will be continuing to enhance client partitioning, evaluating and improving its safety profile as a therapeutic approach, and validating its efficacy in T cell function rescuing in pre-clinical models.