(Research Hub Theater) Manufacture of Hybrid TREG/Th2 Cells That Express Immune Suppressive Molecules and Functionally Suppress Inflammatory CNS Microglial Cells

Background and/or Objectives: Regulatory T-cell therapy in ALS patients mediates an anti-inflammatory effect against microglial cells. In IND-enabling research in preparation for a phase 1 clinical trial in ALS, our goal was to: (1) develop a T cell manufacturing method that resulted in the generation of hybrid T cells that co-expressed both TREG and Th2 transcription factors (FOXP3 and GATA3, respectively), the homing molecule CD103 that facilitates lymphocyte traffic across the blood-brain-barrier, and the anti-inflammatory ATP ecto-nucleotidase molecule CD73; and (2) determine whether such hybrid TREG/Th2 cells could reduce CNS microglial cell inflammation.

Design: Human T cells were manufactured ex vivo with temsirolimus [2 uM), IL-2 [1000 IU/ml] and TGF-B [10 ng/mL]), and IL-4 [1000 IU/mL]) to generate hybrid TREG/Th2 cells.

Setting: Pre-clinical IND enabling research for a phase 1 first-in-human ALS neurological clinical trial

Participants: Normal volunteer samples. Lymphocytes obtained on an IRB-approved protocol

Interventions: N/A

Main Outcome Measures: Intracellular flow cytometry to evaluate cell markers. Surface flow cytometry to evaluate CD103 and CD73 expression. In transwell culture experiments, determine the ability of TREG/Th2 cells to reduce inflammatory cytokine and chemokine secretion from a human CNS microglial cell line (HMC3 cells)

Results: Manufactured T cells expressed FOXP3 (Percent Positive [%+], 33.4 ± 2.0; increased relative to culture input, p< 0.05), increased GATA3 (%+, 40.6 ± 3.6; p< 0.05), increased CD103 (%+; 13.9 ± 2.0; p< 0.05), and CD73 (%+; 25.3 ± 2.6). Addition of TREG/Th2 cells to the HMC3 transwell at a dilute ratio (1:40) reduced inflammatory molecules, including IL-6, which is associated with ALS pathogenesis.

Conclusions: Ex vivo T cell manufacturing can generate hybrid TREG/Th2 cells that show promise for the treatment of ALS as they express immune suppressive molecules and suppress human CNS microglial cells. This research successfully supported an FDA-IND application and initiation of an ongoing phase 1 trial of hybrid TREG/Th2 cells in ALS patients (NCT04220190).