Double activation and low IL-2 manufacturing protocol increase expansion of healthy and patient-derived CAR-Tregs.

Chimeric antigen receptor (CAR)-engineered regulatory T cells (CAR-Tregs) are a promising approach to restore immune tolerance in autoimmune diseases (ADs). However, the low frequency of circulating Tregs, especially in patients with autoimmunity, might limit their applicability as a clinical approach. Thus, an optimal protocol for in vitro expansion of CAR-Tregs would be highly beneficial. We identified three major variables that could affect Treg expansion: the intensity of TCR triggering to activate the cells, the concentration of IL-2, and the number of activation cycles. We combined these variables to compare different CAR-Treg expansion protocols, starting from CD4+CD25+ Tregs transduced with FOXP3 gene and cultured in IL-2 and rapamycin, and we compared them with our standard approach. Two rounds of Treg activation, at a bead-to-cell ratio of 1:1 promoted the optimal expansion of CAR-Tregs, without affecting their phenotype or suppressive function. Dual-activated CAR-Tregs retained their favorable safety profile and reduced the accumulation of activated conventional T cells in organs in a model of xeno-GvHD. When applied to patient-derived Tregs, the dual-activation protocol promoted a robust and higher expansion compared with our standard strategy, while preserving their functionality.