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Updated: Sep 10, 2025
Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice
Published on: June 22, 2016
Detailed analysis of the effects of a NOT gate on activation and growth of T cells
Yuta Ando1, Jingli A Zhang1, Ethan A McLeod1
1A2 Biotherapeutics, Agoura Hills, CA, USA.
Background Aims:
Dual-receptor NOT gates provide a mechanism to target effector cells to antigens that are absent in specific tissues, a situation that occurs frequently in cancer. For example, loss of heterozygosity (LOH) is a genetic event that removes large segments of one or the other homologous chromosome. Roughly 20% of the genes in an average solid tumor undergo LOH by the time of clonal neoplastic expansion, such that these irreversible genetic lesions are present in every cell of the tumor. To exploit this opportunity for selective targeting and other situations that arise in disease, a version of a NOT gate called TmodTM technology has been developed. The Tmod platform incorporates two receptors: an activator based on a CAR or TCR, and a blocker based on the LIR-1 inhibitory receptor.
Methods:
MHC class-I-regulated constructs display robust modularity, functioning well with a variety of activators-both CARs and TCRs-and blocker antigens encoded by different HLA class I alleles. We explore the details of activation, proliferation and cytotoxicity of the Tmod technology, focusing on a HER2 construct, but generalizing the conclusions by experiments with other Tmod constructs.
Results:
We show that Tmod cells exhibit potent, selective tumor-killing even when surrounded by class-I-expressing cells in 3-dimensional spheroids in vitro and in the mouse body. This behavior is largely ligand-dependent, though there are small ligand-independent effects which are mainly ascribed to mechanisms other than the ITIM sequences of the LIR-1 blocker.
Conclusions:
These detailed studies demonstrate that most of Tmod regulation is ligand dependent. Expression levels of the CAR affect activation/proliferation in a LIR-1 NOT gate, but the ITIM signaling module plays only a minor role in ligand-independent activity.
Insights
Dual-receptor NOT gates, like Tmod technology, selectively target cancer cells by exploiting genetic changes such as loss of heterozygosity (LOH). This approach enables potent tumor cell killing while sparing healthy tissues.
Area of Science:
- Immunology
- Oncology
- Genetic Engineering
Background:
- Dual-receptor NOT gates offer a strategy to target cancer cells by exploiting antigens absent in healthy tissues, a common scenario in tumors due to genetic events like loss of heterozygosity (LOH).
- Tmod technology is a novel NOT gate platform integrating an activator (CAR or TCR) and a blocker (LIR-1 inhibitory receptor) for targeted therapy.
Purpose of the Study:
- To investigate the efficacy and regulatory mechanisms of Tmod technology for selective cancer targeting.
- To evaluate the activation, proliferation, and cytotoxicity of Tmod cells in various experimental models.
Main Methods:
- Utilized MHC class-I-regulated constructs with diverse activators (CARs, TCRs) and blocker antigens (HLA class I alleles).
- Focused on a HER2 construct while generalizing findings with other Tmod constructs to explore in vitro 3D spheroids and in vivo mouse models.
Main Results:
- Tmod cells demonstrated potent and selective tumor cell killing in vitro and in vivo, even in the presence of class-I-expressing cells.
- Tumor-killing activity was predominantly ligand-dependent, with minor ligand-independent effects attributed to mechanisms beyond LIR-1 ITIM sequences.
Conclusions:
- Tmod technology's regulation is primarily ligand-dependent.
- CAR expression levels influence Tmod cell activation and proliferation, but the ITIM signaling module has a minimal role in ligand-independent activity.
