Ex vivo activation and fold-expansion of human regulatory T cells negatively correlates with preservation of beta cell function in adolescents with type 1 diabetes

Abstract Type 1 diabetes (T1D) is an autoimmune disease marked by immune destruction of insulin-producing β-cells of the pancreas. In an effort to halt disease progression, autologous polyclonal expanded regulatory T cells (expTregs) were adoptively transferred into adolescents with recent-onset T1D (Sanford/Lisata Therapeutics Trex Phase 2 clinical trial, NCT02691247). Overall, expTreg therapy (1–24×10 6cells/kg) did not preserve residual β-cell function, as measured by change in insulin C-peptide AUC-mean over 2 years compared to placebo (p=0.39) regardless of dose (p=0.27). Consistent with a more activated phenotype, expTregs expressed more CD25, CD38, HLA-DR, and CXCR3 than endogenous Tregs by flow cytometry, and bulk-RNAseq revealed 2377 genes (related to metabolism, cell cycle, and immune response) up-regulated in expTregs compared to endogenous Tregs. In subjects from the high-dose group (>10×10 6cells/kg), there was a transient increase of total Tregs one week after infusion (p=0.003), which had an activated memory phenotype, consistent with infused expTreg accounting for this difference. However, the increase in the activation of Tregs post-treatment was associated with poorer outcome (p=0.03), and the extent of expansion of the expTregs ex vivo (range 24.3–643-fold) negatively correlated with C-peptide change (p=0.01) in treated subjects regardless of Treg dose. Yet, neither purity nor suppressive function correlated with outcome, suggesting differences in endogenous Treg qualities leading to increased expansion may influence outcome. These results suggest that expTreg quantity alone does not influence outcome; instead, both endogenous and expTreg quality may be important factors modulating T1D progression.