FRI616 Combined Elf5a And Notch Inhibition Reprograms The Effector T Cell Fate Decisions Towards Treg Phenotype Without Any Off Target Effect

Abstract

Abstract Disclosure: S. Rafiqi: None. A. Aldasouqi: None. S. faisal: None. S. Qureshi: None. A. Mahmood: None. J.C. Jaume: None. S. Imam: None. We explored immune resetting and plasticity of T effector cells towards T regulatory cells (CD4+CD25+FOXP3+) by elF5A and Notch inhibition. We validated our approach for plasticizing human T effector cells into Treg cells conducted on recent onset latent autoimmune diabetes of adult (LADA) patients (n=3), and nondiabetic patients (n=4) with simultaneous use of GC7 (100 μM) and anti-DLL4 (10 μg/ml). We also investigated the functional stability of plasticized Tregs and compared it with freshly isolated naïve Tregs from the same patient. Tregs were co-cultured with T effector cells and suppression/proliferation was accessed after 5 days. To create an ex-vivo Treg deficient environment, we sorted out CD4+ cells from the PBMCs. CD4+ deficient PBMCs and sorted single positive CD4+CD25- cells were treated with anti-DLL4+GC7+rhGAD65 with and without IL-2 supplementation and were compared with conventional anti-(CD3+CD28) stimulation and absolute control (20%FCS+RPMI). The presence of Treg cells was confirmed by flow cytometry. 30.5±5.1% of CD4 cells were plasticized into Tregs in the treatment group supplemented with IL-2 while only 18.8±4.6 % were plasticized in treatment group without IL-2 supplementation. Also, 11.2±3.1% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 6.19±2.4% in absolute control group. Similarly, 13±4.3% of CD8 cells had plasticized into Tregs in treatment group supplemented with IL-2 while only 11.5±6.7% plasticized in treatment group without IL-2 supplementation. 14.3±5.8% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 12.4±6.3% in absolute control group. Interestingly, the plasticized Tregs from LADA patients were more robust than those of non-diabetic patients at a 1:2 ratio (Treg:Tresp). Furthermore, the plasticized Tregs had comparable suppressive capacity over Tresp (CD4+CD25-) cells as that of freshly isolated naïve human Tregs. To investigate any off-target effects of (GC7 and anti-DLL4), live, dead, and apoptotic cell count at 24, 48, 96 hours, and 7 days post-treatment using dead cell apoptosis kit (Invitrogen). In our study, there was no significant difference between treatment and control groups in terms of live, dead, and apoptotic cells at different time intervals. On the 7th day of culture 64.06±8.66% and 68.88±6.15% of cells were live in treatment and absolute control groups respectively while 7.24±1.59% and 10.0±2.02% cells were found apoptotic respectively. Therefore, we define the reprogramming of T effector cells towards Treg cells with functional suppressive capacity using GC7 and anti-DLL4 without any off-target effect (toxicity). The present study expands the concept of T cell plasticity and establishes the functional stability of plasticized T cells that can accelerate pre-clinical trials for therapeutic use in T1D and as an adjunct to immunotherapies. Presentation: Friday, June 16, 2023