GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response.

RIG-I recognizes viral dsRNA and activates a cell-autonomous antiviral response. Upon stimulation, it triggers a signaling cascade leading to the production of type I and III IFNs. IFNs are secreted and signal to elicit the expression of IFN-stimulated genes, establishing an antiviral state of the cell. The topology of this pathway has been studied intensively, however, its exact dynamics are less understood. Here, we employed electroporation to synchronously activate RIG-I, enabling us to characterize cell-intrinsic innate immune signaling at a high temporal resolution. Employing IFNAR1/IFNLR-deficient cells, we could differentiate primary RIG-I signaling from secondary signaling downstream of the IFN receptors. Based on these data, we developed a comprehensive mathematical model capable of simulating signaling downstream of dsRNA recognition by RIG-I and the feedback and signal amplification by IFN. We further investigated the impact of viral antagonists on signaling dynamics. Our work provides a comprehensive insight into the signaling events that occur early upon virus infection and opens new avenues to study and disentangle the complexity of the host-virus interface.

Mechanistic modeling explains the dsRNA length-dependent activation of the RIG-I mediated immune response.

In cell-intrinsic antiviral immunity, cytoplasmic receptors such as retinoic acid-inducible gene I (RIG-I) detect viral double-stranded RNA (dsRNA) and trigger a signaling cascade activating the interferon (IFN) system. This leads to the transcription of hundreds of interferon-stimulated genes (ISGs) with a wide range of antiviral effects. This recognition of dsRNA not only has to be very specific to discriminate foreign from self but also highly sensitive to detect even very low numbers of pathogenic dsRNA molecules. Previous work indicated an influence of the dsRNA length on the binding behavior of RIG-I and its potential to elicit antiviral signaling. However, the molecular mechanisms behind the binding process are still under debate. We compare two hypothesized RIG-I binding mechanisms by translating them into mathematical models and analyzing their potential to describe published experimental data. The models consider the length of the dsRNA as well as known RIG-I binding motifs and describe RIG-I pathway activation after stimulation with dsRNA. We show that internal RIG-I binding sites in addition to cooperative RIG-I oligomerization are essential to describe the experimentally observed RIG-I binding behavior and immune response activation for different dsRNA lengths and concentrations. The combination of RIG-I binding to internal sites on the dsRNA and cooperative oligomerization compensates for a lack of high-affinity binding motifs and triggers a strong antiviral response for long dsRNAs. Model analysis reveals dsRNA length-dependency as a potential mechanism to discriminate between different types of dsRNAs: It allows for sensitive detection of small numbers of long dsRNAs, a typical by-product of viral replication, while ensuring tolerance against non-harming small dsRNAs.